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Sample records for recognition complex subunit

  1. A subunit of the oligosaccharyltransferase complex is required for interspecific gametophyte recognition in Arabidopsis

    PubMed Central

    Müller, Lena M.; Lindner, Heike; Pires, Nuno D.; Gagliardini, Valeria; Grossniklaus, Ueli

    2016-01-01

    Species-specific gamete recognition is a key premise to ensure reproductive success and the maintenance of species boundaries. During plant pollen tube (PT) reception, gametophyte interactions likely allow the species-specific recognition of signals from the PT (male gametophyte) by the embryo sac (female gametophyte), resulting in PT rupture, sperm release, and double fertilization. This process is impaired in interspecific crosses between Arabidopsis thaliana and related species, leading to PT overgrowth and a failure to deliver the sperm cells. Here we show that ARTUMES (ARU) specifically regulates the recognition of interspecific PTs in A. thaliana. ARU, identified in a genome-wide association study (GWAS), exclusively influences interspecific—but not intraspecific—gametophyte interactions. ARU encodes the OST3/6 subunit of the oligosaccharyltransferase complex conferring protein N-glycosylation. Our results suggest that glycosylation patterns of cell surface proteins may represent an important mechanism of gametophyte recognition and thus speciation. PMID:26964640

  2. Subsets of human origin recognition complex (ORC) subunits are expressed in non-proliferating cells and associate with non-ORC proteins.

    PubMed

    Thome, K C; Dhar, S K; Quintana, D G; Delmolino, L; Shahsafaei, A; Dutta, A

    2000-11-10

    The origin recognition complex (ORC) in yeast is a complex of six tightly associated subunits essential for the initiation of DNA replication. Human ORC subunits are nuclear in proliferating cells and in proliferative tissues like the testis, consistent with a role of human ORC in DNA replication. Orc2, Orc3, and Orc5 also are detected in non-proliferating cells like cardiac myocytes, adrenal cortical cells, and neurons, suggesting an additional role of these proteins in non-proliferating cells. Although Orc2-5 co-immunoprecipitate with each other under mild extraction conditions, a holo complex of the subunits is difficult to detect. When extracted under more stringent extraction conditions, several of the subunits co-immunoprecipitate with stoichiometric amounts of other unidentified proteins but not with any of the known ORC subunits. The variation in abundance of individual ORC subunits (relative to each other) in several tissues, expression of some subunits in non-proliferating tissues, and the absence of a stoichiometric complex of all the subunits in cell extracts indicate that subunits of human ORC in somatic cells might have activities independent of their role as a six subunit complex involved in replication initiation. Finally, all ORC subunits remain consistently nuclear, and Orc2 is consistently phosphorylated through all stages of the cell cycle, whereas Orc1 is selectively phosphorylated in mitosis.

  3. Genetic interaction of an origin recognition complex subunit and the Polycomb group gene MEDEA during seed development.

    PubMed

    Collinge, Margaret A; Spillane, Charles; Köhler, Claudia; Gheyselinck, Jacqueline; Grossniklaus, Ueli

    2004-04-01

    The eukaryotic origin recognition complex (ORC) is made up of six subunits and functions in nuclear DNA replication, chromatin structure, and gene silencing in both fungi and metazoans. We demonstrate that disruption of a plant ORC subunit homolog, AtORC2 of Arabidopsis (Arabidopsis thaliana), causes a zygotic lethal mutant phenotype (orc2). Seeds of orc2 abort early, typically producing embryos with up to eight cells. Nuclear division in the endosperm is arrested at an earlier developmental stage: only approximately four nuclei are detected in orc2 endosperm. The endosperm nuclei in orc2 are dramatically enlarged, a phenotype that is most similar to class B titan mutants, which include mutants in structural maintenance of chromosomes (SMC) cohesins. The highest levels of ORC2 gene expression were found in preglobular embryos, coinciding with the stage at which homozygous orc2 mutant seeds arrest. The homologs of the other five Arabidopsis ORC subunits are also expressed at this developmental stage. The orc2 mutant phenotype is partly suppressed by a mutation in the Polycomb group gene MEDEA. In double mutants between orc2 and medea (mea), orc2 homozygotes arrest later with a phenotype intermediate between those of mea and orc2 single mutants. Either alterations in chromatin structure or the release of cell cycle checkpoints by the mea mutation may allow more cell and nuclear divisions to occur in orc2 homozygous seeds.

  4. NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes.

    PubMed

    Smrcka, Alan V; Kichik, Nessim; Tarragó, Teresa; Burroughs, Michael; Park, Min-Sun; Itoga, Nathan K; Stern, Harry A; Willardson, Barry M; Giralt, Ernest

    2010-01-12

    G-protein betagamma (Gbetagamma) subunits interact with a wide range of molecular partners including: G(alpha) subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by Gbetagamma, a method for site-specific (15)N-labeling of Gbeta-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of (15)N-Trp Gbetagamma identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for GbetaW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GbetaW99 is highly mobile on the nanosecond timescale in comparison with other Gbeta tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GbetaW99 and GbetaW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of G(alpha)(i1)-GDP to Gbetagamma had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that Gbetagamma subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.

  5. Characterization of Choristoneura fumiferana genes of the sixth subunit of the origin recognition complex: CfORC6.

    PubMed

    Wang, Xaiochun; Carstens, Eric B; Feng, Qili

    2006-11-30

    A new protein was cloned and identified as the sixth subunit of Choristoneura fumiferana origin recognition complex (CfORC6). The newly identified 43 kDa protein CfORC6 is much bigger than DmORC6 (25.7 kDa) and HsORC6 (28.1 kDa), though itos 23.85% identical to DmORC6 and 23.81% identical to HsORC6. Although the molecular weight of CfORC6 is close to ScORc6 (50 kDa), CfORC6 is only 14.03% identical to ScORC6. By alignment, it was found that the N-terminal of CfORC6 has about 30% identities with other ORC6s, but about 100aa of C-terminal of CfORC6 has no identity with other ORC6s. Like ScORC6, CfORC6 has many potential phosphorylation sites, (S/T)PXK. Like DmORC6, CfORC6 has leucine-rich region in the relevant site. Northern Blot showed that CfORC6 mRNA is about 2,000nt. Southern Blot confirmed that there is one copy of CfORC6 gene in spruce budworm genome. Western blot showed that infection of Cf124T cells with CfMNPV didnot affect the expression levels of CfORC6, at least up to 26 hr post infection.

  6. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    NASA Technical Reports Server (NTRS)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  7. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    NASA Technical Reports Server (NTRS)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  8. Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex*

    PubMed Central

    Mardones, Gonzalo A.; Burgos, Patricia V.; Lin, Yimo; Kloer, Daniel P.; Magadán, Javier G.; Hurley, James H.; Bonifacino, Juan S.

    2013-01-01

    Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides. PMID:23404500

  9. Structural basis for the recognition of tyrosine-based sorting signals by the μ3A subunit of the AP-3 adaptor complex.

    PubMed

    Mardones, Gonzalo A; Burgos, Patricia V; Lin, Yimo; Kloer, Daniel P; Magadán, Javier G; Hurley, James H; Bonifacino, Juan S

    2013-03-29

    Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14-19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.

  10. Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome.

    PubMed

    Bicknell, Louise S; Walker, Sarah; Klingseisen, Anna; Stiff, Tom; Leitch, Andrea; Kerzendorfer, Claudia; Martin, Carol-Anne; Yeyati, Patricia; Al Sanna, Nouriya; Bober, Michael; Johnson, Diana; Wise, Carol; Jackson, Andrew P; O'Driscoll, Mark; Jeggo, Penny A

    2011-02-27

    Studies into disorders of extreme growth failure (for example, Seckel syndrome and Majewski osteodysplastic primordial dwarfism type II) have implicated fundamental cellular processes of DNA damage response signaling and centrosome function in the regulation of human growth. Here we report that mutations in ORC1, encoding a subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. We establish that these mutations disrupt known ORC1 functions including pre-replicative complex formation and origin activation. ORC1 deficiency perturbs S-phase entry and S-phase progression. Additionally, we show that Orc1 depletion in zebrafish is sufficient to markedly reduce body size during rapid embryonic growth. Our data suggest a model in which ORC1 mutations impair replication licensing, slowing cell cycle progression and consequently impeding growth during development, particularly at times of rapid proliferation. These findings establish a novel mechanism for the pathogenesis of microcephalic dwarfism and show a surprising but important developmental impact of impaired origin licensing.

  11. Structural basis for the recognition of methylated histone H3K36 by the Eaf3 subunit of histone deacetylase complex Rpd3S.

    PubMed

    Xu, Chao; Cui, Gaofeng; Botuyan, Maria Victoria; Mer, Georges

    2008-11-12

    Deacetylation of nucleosomes by the Rpd3S histone deacetylase along the path of transcribing RNA polymerase II regulates access to DNA, contributing to faithful gene transcription. The association of Rpd3S with chromatin requires its Eaf3 subunit, which binds histone H3 methylated at lysine 36 (H3K36). Eaf3 is also part of NuA4 acetyltransferase that recognizes methylated H3K4. Here we show that Eaf3 in Saccharomyces cerevisiae contains a chromo barrel-related domain that binds methylated peptides, including H3K36 and H3K4, with low specificity and millimolar-range affinity. Nuclear magnetic resonance structure determination of Eaf3 bound to methylated H3K36 was accomplished by engineering a linked Eaf3-H3K36 molecule with a chemically incorporated methyllysine analog. Our study uncovers the molecular details of Eaf3-methylated H3K36 complex formation, and suggests that, in the cell, Eaf3 can only function within a framework of combinatorial interactions. This work also provides a general method for structure determination of low-affinity protein complexes implicated in methyllysine recognition.

  12. Complex Event Recognition Architecture

    NASA Technical Reports Server (NTRS)

    Fitzgerald, William A.; Firby, R. James

    2009-01-01

    Complex Event Recognition Architecture (CERA) is the name of a computational architecture, and software that implements the architecture, for recognizing complex event patterns that may be spread across multiple streams of input data. One of the main components of CERA is an intuitive event pattern language that simplifies what would otherwise be the complex, difficult tasks of creating logical descriptions of combinations of temporal events and defining rules for combining information from different sources over time. In this language, recognition patterns are defined in simple, declarative statements that combine point events from given input streams with those from other streams, using conjunction, disjunction, and negation. Patterns can be built on one another recursively to describe very rich, temporally extended combinations of events. Thereafter, a run-time matching algorithm in CERA efficiently matches these patterns against input data and signals when patterns are recognized. CERA can be used to monitor complex systems and to signal operators or initiate corrective actions when anomalous conditions are recognized. CERA can be run as a stand-alone monitoring system, or it can be integrated into a larger system to automatically trigger responses to changing environments or problematic situations.

  13. Acute reduction of an origin recognition complex (ORC) subunit in human cells reveals a requirement of ORC for Cdk2 activation.

    PubMed

    Machida, Yuichi J; Teer, Jamie K; Dutta, Anindya

    2005-07-29

    The origin recognition complex (ORC) is involved in formation of prereplicative complexes (pre-RCs) on replication origins in the G1 phase. At the G1/S transition, elevated cyclin E-CDK2 activity triggers 1DNA replication to enter S phase. The CDK cycle works as an engine that drives progression of cell cycle events by successive activation of different types of cyclin-CDK. However, how the CDK cycle is coordinated with replication initiation remains elusive. Here we report that acute depletion of ORC2 by RNA interference (RNAi) arrests cells with low cyclin E-CDK2 activity. This result suggests that loss of a replication initiation protein prevents progression of the CDK cycle in G1. p27 and p21 proteins accumulate following ORC2 RNAi and are required for the CDK2 inhibition. Restoration of CDK activity by co-depletion of p27 and p21 allows many ORC2-depleted cells to enter S phase and go on to mitosis. However, in some cells the release of the CDK2 block caused catastrophic events like apoptosis. Therefore, the CDK2 inhibition observed following ORC2 RNAi seems to protect cells from premature S phase entry and crisis in DNA replication. These results demonstrate an unexpected role of ORC2 in CDK2 activation, a linkage that could be important for maintaining genomic stability.

  14. The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs.

    PubMed Central

    Bovia, F; Fornallaz, M; Leffers, H; Strub, K

    1995-01-01

    The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism. Images PMID:7542942

  15. The TCP1γ subunit of Leishmania donovani forms a biologically active homo-oligomeric complex.

    PubMed

    Bhaskar; Mitra, Kalyan; Kuldeep, Jitendra; Siddiqi, Mohammad Imran; Goyal, Neena

    2015-12-01

    Chaperonins are a class of molecular chaperons that encapsulate nascent or stress-denatured proteins and assist their intracellular assembly and folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP1 ring complex is a hetero-oligomeric complex comprising two rings, each formed of eight subunits that may have distinct substrate recognition and ATP hydrolysis properties. In Leishmania, only the TCP1γ subunit has been cloned and characterized. It exhibited differential expression at various growth stages of promastigotes. In the present study, we expressed the TCP1γ subunit in Escherichia coli to investigate whether it forms chaperonin-like complexes and plays a role in protein folding. LdTCP1γ formed high-molecular-weight complexes within E. coli cells as well as in Leishmania cell lysates. The recombinant protein is arranged into two back-to-back rings of seven subunits each, as predicted by homology modelling and observed by negative staining electron microscopy. This morphology is consistent with that of the oligomeric double-ring group I chaperonins found in mitochondria. The LdTCP1γ homo-oligomeric complex hydrolysed ATP, and was active as assayed by luciferase refolding. Thus, the homo-oligomer performs chaperonin reactions without partner subunit(s). Further, co-immunoprecipitation studies revealed that LdTCP1γ interacts with actin and tubulin proteins, suggesting that the complex may have a role in maintaining the structural dynamics of the cytoskeleton of parasites.

  16. Deficiency of subunits of Complex I and mitochondrial encephalomyopathy.

    PubMed

    Ichiki, T; Tanaka, M; Nishikimi, M; Suzuki, H; Ozawa, T; Kobayashi, M; Wada, Y

    1988-03-01

    Enzymic activities of the respiratory chain and content of immunochemically detectable subunits in NADH-ubiquinone oxidoreductase (Complex I) were measured in mitochondria from the skeletal muscles of 4 patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). The rotenone-sensitive NADH-cytochrome c reductase activity was extremely decreased, ranging from 0% to 27% of the control value. In all patients, the content of subunits of Complex I was also reduced in parallel with the rotenone-sensitive NADH-cytochrome c reductase activity. It is suggested that the variation in the degree of deficiency of Complex I subunits could explain the clinical heterogeneity of patients with MELAS.

  17. Structural Mechanism for Cargo Recognition by the Retromer Complex.

    PubMed

    Lucas, María; Gershlick, David C; Vidaurrazaga, Ander; Rojas, Adriana L; Bonifacino, Juan S; Hierro, Aitor

    2016-12-01

    Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here, we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3, and cargo that couple signal-recognition to membrane recruitment.

  18. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  19. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

    PubMed

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B; Webb, Kristofor; Bennett, Eric J; Vinterbo, Staal; Potter, Clinton S; Carragher, Bridget; Joazeiro, Claudio A P

    2014-11-11

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

  20. Structure of the archaeal Cascade subunit Csa5: relating the small subunits of CRISPR effector complexes.

    PubMed

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F; Naismith, James H

    2013-05-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.

  1. Understanding Molecular Recognition by G protein βγ Subunits on the Path to Pharmacological Targeting

    PubMed Central

    Lin, Yuan

    2011-01-01

    Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transduce extracellular signals via G-protein-coupled receptors to modulate many important intracellular responses. The Gβγ subunits hold a central position in this signaling system and have been implicated in multiple aspects of physiology and the pathophysiology of disease. The Gβ subunit belongs to a large family of WD40 repeat proteins with a circular β-bladed propeller structure. This structure allows Gβγ to interact with a broad range of proteins to play diverse roles. How Gβγ interacts with and regulates such a wide variety of partners yet maintains specificity is an interesting problem in protein-protein molecular recognition in signal transduction, where signal transfer by proteins is often driven by modular conserved recognition motifs. Evidence has accumulated that one mechanism for Gβγ multitarget recognition is through an intrinsically flexible protein surface or “hot spot” that accommodates multiple modes of binding. Because each target has a unique recognition mode for Gβγ subunits, it suggests that these interactions could be selectively manipulated with small molecules, which could have significant therapeutic potential. PMID:21737569

  2. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

    PubMed Central

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2011-01-01

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  3. Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits.

    PubMed

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2011-06-03

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

  4. Mcm subunits can assemble into two different active unwinding complexes.

    PubMed

    Kanter, Diane M; Bruck, Irina; Kaplan, Daniel L

    2008-11-07

    The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.

  5. Energetics of codon-anticodon recognition on the small ribosomal subunit.

    PubMed

    Almlöf, Martin; Andér, Martin; Aqvist, Johan

    2007-01-09

    Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

  6. Targeting signals and subunit interactions in coated vesicle adaptor complexes

    PubMed Central

    1995-01-01

    There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma- adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane. PMID:7593184

  7. The role of TcdB and TccC subunits in secretion of the Photorhabdus Tcd toxin complex.

    PubMed

    Yang, Guowei; Waterfield, Nicholas R

    2013-01-01

    The Toxin Complex (TC) is a large multi-subunit toxin encoded by a range of bacterial pathogens. The best-characterized examples are from the insect pathogens Photorhabdus, Xenorhabdus and Yersinia. They consist of three large protein subunits, designated A, B and C that assemble in a 5∶1∶1 stoichiometry. Oral toxicity to a range of insects means that some have the potential to be developed as pest control technology. The three subunit proteins do not encode any recognisable export sequences and as such little progress has been made in understanding their secretion. We have developed heterologous TC production and secretion models in E. coli and used them to ascribe functions to different domains of the crucial B+C sub-complex. We have determined that the B and C subunits use a secretion mechanism that is either encoded by the proteins themselves or employ an as yet undefined system common to laboratory strains of E. coli. We demonstrate that both the N-terminal domains of the B and C subunits are required for secretion of the whole complex. We propose a model whereby the N-terminus of the C-subunit toxin exports the B+C sub-complex across the inner membrane while that of the B-subunit allows passage across the outer membrane. We also demonstrate that even in the absence of the B-subunit, that the C-subunit can also facilitate secretion of the larger A-subunit. The recognition of this novel export system is likely to be of importance to future protein secretion studies. Finally, the identification of homologues of B and C subunits in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggests that these toxins are likely to be important in a range of different hosts, including man.

  8. The Role of TcdB and TccC Subunits in Secretion of the Photorhabdus Tcd Toxin Complex

    PubMed Central

    Yang, Guowei; Waterfield, Nicholas R.

    2013-01-01

    The Toxin Complex (TC) is a large multi-subunit toxin encoded by a range of bacterial pathogens. The best-characterized examples are from the insect pathogens Photorhabdus, Xenorhabdus and Yersinia. They consist of three large protein subunits, designated A, B and C that assemble in a 5∶1∶1 stoichiometry. Oral toxicity to a range of insects means that some have the potential to be developed as pest control technology. The three subunit proteins do not encode any recognisable export sequences and as such little progress has been made in understanding their secretion. We have developed heterologous TC production and secretion models in E. coli and used them to ascribe functions to different domains of the crucial B+C sub-complex. We have determined that the B and C subunits use a secretion mechanism that is either encoded by the proteins themselves or employ an as yet undefined system common to laboratory strains of E. coli. We demonstrate that both the N-terminal domains of the B and C subunits are required for secretion of the whole complex. We propose a model whereby the N-terminus of the C-subunit toxin exports the B+C sub-complex across the inner membrane while that of the B-subunit allows passage across the outer membrane. We also demonstrate that even in the absence of the B-subunit, that the C-subunit can also facilitate secretion of the larger A-subunit. The recognition of this novel export system is likely to be of importance to future protein secretion studies. Finally, the identification of homologues of B and C subunits in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggests that these toxins are likely to be important in a range of different hosts, including man. PMID:24098116

  9. CMF70 is a subunit of the dynein regulatory complex

    PubMed Central

    Kabututu, Zakayi P.; Thayer, Michelle; Melehani, Jason H.; Hill, Kent L.

    2010-01-01

    Flagellar motility drives propulsion of several important pathogens and is essential for human development and physiology. Motility of the eukaryotic flagellum requires coordinate regulation of thousands of dynein motors arrayed along the axoneme, but the proteins underlying dynein regulation are largely unknown. The dynein regulatory complex, DRC, is recognized as a focal point of axonemal dynein regulation, but only a single DRC subunit, trypanin/PF2, is currently known. The component of motile flagella 70 protein, CMF70, is broadly and uniquely conserved among organisms with motile flagella, suggesting a role in axonemal motility. Here we demonstrate that CMF70 is part of the DRC from Trypanosoma brucei. CMF70 is located along the flagellum, co-sediments with trypanin in sucrose gradients and co-immunoprecipitates with trypanin. RNAi knockdown of CMF70 causes motility defects in a wild-type background and suppresses flagellar paralysis in cells with central pair defects, thus meeting the functional definition of a DRC subunit. Trypanin and CMF70 are mutually conserved in at least five of six extant eukaryotic clades, indicating that the DRC was probably present in the last common eukaryotic ancestor. We have identified only the second known subunit of this ubiquitous dynein regulatory system, highlighting the utility of combined genomic and functional analyses for identifying novel subunits of axonemal sub-complexes. PMID:20876659

  10. Extricating Manual and Non-Manual Features for Subunit Level Medical Sign Modelling in Automatic Sign Language Classification and Recognition.

    PubMed

    R, Elakkiya; K, Selvamani

    2017-09-22

    Subunit segmenting and modelling in medical sign language is one of the important studies in linguistic-oriented and vision-based Sign Language Recognition (SLR). Many efforts were made in the precedent to focus the functional subunits from the view of linguistic syllables but the problem is implementing such subunit extraction using syllables is not feasible in real-world computer vision techniques. And also, the present recognition systems are designed in such a way that it can detect the signer dependent actions under restricted and laboratory conditions. This research paper aims at solving these two important issues (1) Subunit extraction and (2) Signer independent action on visual sign language recognition. Subunit extraction involved in the sequential and parallel breakdown of sign gestures without any prior knowledge on syllables and number of subunits. A novel Bayesian Parallel Hidden Markov Model (BPaHMM) is introduced for subunit extraction to combine the features of manual and non-manual parameters to yield better results in classification and recognition of signs. Signer independent action aims in using a single web camera for different signer behaviour patterns and for cross-signer validation. Experimental results have proved that the proposed signer independent subunit level modelling for sign language classification and recognition has shown improvement and variations when compared with other existing works.

  11. The centromere-kinetochore complex: a repeat subunit model

    PubMed Central

    1991-01-01

    The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species. PMID:1828250

  12. Complex Wavelet Transform-Based Face Recognition

    NASA Astrophysics Data System (ADS)

    Eleyan, Alaa; Özkaramanli, Hüseyin; Demirel, Hasan

    2009-12-01

    Complex approximately analytic wavelets provide a local multiscale description of images with good directional selectivity and invariance to shifts and in-plane rotations. Similar to Gabor wavelets, they are insensitive to illumination variations and facial expression changes. The complex wavelet transform is, however, less redundant and computationally efficient. In this paper, we first construct complex approximately analytic wavelets in the single-tree context, which possess Gabor-like characteristics. We, then, investigate the recently developed dual-tree complex wavelet transform (DT-CWT) and the single-tree complex wavelet transform (ST-CWT) for the face recognition problem. Extensive experiments are carried out on standard databases. The resulting complex wavelet-based feature vectors are as discriminating as the Gabor wavelet-derived features and at the same time are of lower dimension when compared with that of Gabor wavelets. In all experiments, on two well-known databases, namely, FERET and ORL databases, complex wavelets equaled or surpassed the performance of Gabor wavelets in recognition rate when equal number of orientations and scales is used. These findings indicate that complex wavelets can provide a successful alternative to Gabor wavelets for face recognition.

  13. Crystal Structure of the Eukaryotic Origin Recognition Complex

    PubMed Central

    Bleichert, Franziska; Botchan, Michael R.; Berger, James M.

    2015-01-01

    Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. The 3.5 Å resolution crystal structure of Drosophila ORC reveals that the 270 kDa initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ ATPase folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident, including highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighboring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate a ~20 Å wide channel in the center of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the MCM2-7 complex during replicative helicase loading; however, an observed >90° out-of-plane rotation for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions. PMID:25762138

  14. Catalytic subunit stoichiometry within the cellulose synthase complex.

    PubMed

    Gonneau, Martine; Desprez, Thierry; Guillot, Alain; Vernhettes, Samantha; Höfte, Herman

    2014-12-01

    Cellulose synthesis is driven by large plasma membrane-inserted protein complexes, which in plants have 6-fold symmetry. In Arabidopsis (Arabidopsis thaliana), functional cellulose synthesis complexes (CSCs) are composed of at least three different cellulose synthase catalytic subunits (CESAs), but the actual ratio of the CESA isoforms within the CSCs remains unresolved. In this work, the stoichiometry of the CESAs in the primary cell wall CSC was determined, after elimination of CESA redundancy in a mutant background, by coimmunoprecipitation and mass spectrometry using label-free quantitative methods. Based on spectral counting, we show that CESA1, CESA3, and CESA6 are present in a 1:1:1 molecular ratio. © 2014 American Society of Plant Biologists. All Rights Reserved.

  15. Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments

    PubMed Central

    Zhu, Jiapeng; King, Martin S.; Yu, Minmin; Klipcan, Liron; Leslie, Andrew G. W.; Hirst, Judy

    2015-01-01

    Mitochondrial complex I (proton-pumping NADH:ubiquinone oxidoreductase) is an essential respiratory enzyme. Mammalian complex I contains 45 subunits: 14 conserved “core” subunits and 31 “supernumerary” subunits. The structure of Bos taurus complex I, determined to 5-Å resolution by electron cryomicroscopy, described the structure of the mammalian core enzyme and allowed the assignment of 14 supernumerary subunits. Here, we describe the 6.8-Å resolution X-ray crystallography structure of subcomplex Iβ, a large portion of the membrane domain of B. taurus complex I that contains two core subunits and a cohort of supernumerary subunits. By comparing the structures and composition of subcomplex Iβ and complex I, supported by comparisons with Yarrowia lipolytica complex I, we propose assignments for eight further supernumerary subunits in the structure. Our new assignments include two CHCH-domain containing subunits that contain disulfide bridges between CX9C motifs; they are processed by the Mia40 oxidative-folding pathway in the intermembrane space and probably stabilize the membrane domain. We also assign subunit B22, an LYR protein, to the matrix face of the membrane domain. We reveal that subunit B22 anchors an acyl carrier protein (ACP) to the complex, replicating the LYR protein–ACP structural module that was identified previously in the hydrophilic domain. Thus, we significantly extend knowledge of how the mammalian supernumerary subunits are arranged around the core enzyme, and provide insights into their roles in biogenesis and regulation. PMID:26371297

  16. Unique subunit packing in mycobacterial nanoRNase leads to alternate substrate recognitions in DHH phosphodiesterases

    PubMed Central

    Srivastav, Rajpal; Kumar, Dilip; Grover, Amit; Singh, Ajit; Manjasetty, Babu A.; Sharma, Rakesh; Taneja, Bhupesh

    2014-01-01

    DHH superfamily includes RecJ, nanoRNases (NrnA), cyclic nucleotide phosphodiesterases and pyrophosphatases. In this study, we have carried out in vitro and in vivo investigations on the bifunctional NrnA-homolog from Mycobacterium smegmatis, MSMEG_2630. The crystal structure of MSMEG_2630 was determined to 2.2-Å resolution and reveals a dimer consisting of two identical subunits with each subunit folding into an N-terminal DHH domain and a C-terminal DHHA1 domain. The overall structure and fold of the individual domains is similar to other members of DHH superfamily. However, MSMEG_2630 exhibits a distinct quaternary structure in contrast to other DHH phosphodiesterases. This novel mode of subunit packing and variations in the linker region that enlarge the domain interface are responsible for alternate recognitions of substrates in the bifunctional nanoRNases. MSMEG_2630 exhibits bifunctional 3′-5′ exonuclease [on both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) substrates] as well as CysQ-like phosphatase activity (on pAp) in vitro with a preference for nanoRNA substrates over single-stranded DNA of equivalent lengths. A transposon disruption of MSMEG_2630 in M. smegmatis causes growth impairment in the presence of various DNA-damaging agents. Further phylogenetic analysis and genome organization reveals clustering of bacterial nanoRNases into two distinct subfamilies with possible role in transcriptional and translational events during stress. PMID:24878921

  17. Multipoint molecular recognition within a calix[6]arene funnel complex

    PubMed Central

    Coquière, David; de la Lande, Aurélien; Martí, Sergio; Parisel, Olivier; Prangé, Thierry; Reinaud, Olivia

    2009-01-01

    A multipoint recognition system based on a calix[6]arene is described. The calixarene core is decorated on alternating aromatic subunits by 3 imidazole arms at the small rim and 3 aniline groups at the large rim. This substitution pattern projects the aniline nitrogens toward each other when Zn(II) binds at the Tris-imidazole site or when a proton binds at an aniline. The XRD structure of the monoprotonated complex having an acetonitrile molecule bound to Zn(II) in the cavity revealed a constrained geometry at the metal center reminiscent of an entatic state. Computer modeling suggests that the aniline groups behave as a tritopic monobasic site in which only 1 aniline unit is protonated and interacts with the other 2 through strong hydrogen bonding. The metal complex selectively binds a monoprotonated diamine vs. a monoamine through multipoint recognition: coordination to the metal ion at the small rim, hydrogen bonding to the calix-oxygen core, CH/π interaction within the cavity's aromatic walls, and H-bonding to the anilines at the large rim. PMID:19237564

  18. Accessory subunits are integral for assembly and function of human mitochondrial complex I.

    PubMed

    Stroud, David A; Surgenor, Elliot E; Formosa, Luke E; Reljic, Boris; Frazier, Ann E; Dibley, Marris G; Osellame, Laura D; Stait, Tegan; Beilharz, Traude H; Thorburn, David R; Salim, Agus; Ryan, Michael T

    2016-10-06

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

  19. The program complex for vocal recognition

    NASA Astrophysics Data System (ADS)

    Konev, Anton; Kostyuchenko, Evgeny; Yakimuk, Alexey

    2017-01-01

    This article discusses the possibility of applying the algorithm of determining the pitch frequency for the note recognition problems. Preliminary study of programs-analogues were carried out for programs with function “recognition of the music”. The software package based on the algorithm for pitch frequency calculation was implemented and tested. It was shown that the algorithm allows recognizing the notes in the vocal performance of the user. A single musical instrument, a set of musical instruments, and a human voice humming a tune can be the sound source. The input file is initially presented in the .wav format or is recorded in this format from a microphone. Processing is performed by sequentially determining the pitch frequency and conversion of its values to the note. According to test results, modification of algorithms used in the complex was planned.

  20. The multicoloured world of promoter recognition complexes

    PubMed Central

    Müller, Ferenc; Tora, Làszlò

    2004-01-01

    The expression pattern of regulated genes changes dynamically depending on the developmental stage and the differentiation state of the cell. Transcription factors regulate cellular events at the gene expression level by communicating signals to the general transcription machinery that forms a preinitiation complex (PIC) at class II core promoters. Recent data strongly suggest that PICs are composed of different sets of factors at distinct promoters, reflecting the spatiotemporal profile of gene expression in multicellular organisms. Thus, today it is important to ask the question: how universal are the promoter recognition factors? This review will focus on findings that support the new idea that core promoter recognition by distinct factors is an additional level of transcriptional regulation and that this step is developmentally regulated. PMID:14685269

  1. Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

    PubMed Central

    Shi, Jiong; Friedman, David B.

    2013-01-01

    The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface. PMID:23785198

  2. Functional architecture of the retromer cargo-recognition complex

    PubMed Central

    Hierro, Aitor; Rojas, Adriana L.; Rojas, Raul; Murthy, Namita; Effantin, Grégory; Kajava, Andrey V.; Steven, Alasdair C.; Bonifacino, Juan S.; Hurley, James H.

    2008-01-01

    The retromer complex 1, 2 is required for the sorting of acid hydrolases to lysosomes 3-7, transcytosis of the polymeric Ig receptor 8, Wnt gradient formation 9, 10, iron transporter recycling 11, and processing of the amyloid precursor protein 12. Human retromer consists of two smaller complexes, the cargo recognition Vps26:Vps29:Vps35 heterotrimer, and a membrane-targeting heterodimer or homodimer of SNX1 and/or SNX2 13. The crystal structure of a Vps29:Vps35 subcomplex shows how the metallophosphoesterase-fold subunit Vps29 14, 15 acts as a scaffold for the C-terminal half of Vps35. Vps35 forms a horseshoe-shaped right-handed α-helical solenoid whose concave face completely covers the metal-binding site of Vps29 and whose convex face exposes a series of hydrophobic interhelical grooves. Electron microscopy shows that the intact Vps26:Vps29:Vps35 complex is a stick-shaped, somewhat flexible, structure, ∼ 21 nm long. A hybrid structural model derived from crystal structures, electron microscopy, interaction studies, and bioinformatics shows that the α-solenoid fold extends the full length of Vps35, and that Vps26 is bound at the opposite end from Vps29. This extended structure presents multiple binding sites for the SNX complex and receptor cargo, and appears capable of flexing to conform to curved vesicular membranes. PMID:17891154

  3. Functional architecture of the retromer cargo-recognition complex.

    PubMed

    Hierro, Aitor; Rojas, Adriana L; Rojas, Raul; Murthy, Namita; Effantin, Grégory; Kajava, Andrey V; Steven, Alasdair C; Bonifacino, Juan S; Hurley, James H

    2007-10-25

    The retromer complex is required for the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric immunoglobulin receptor, Wnt gradient formation, iron transporter recycling and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes: the cargo recognition VPS26-VPS29-VPS35 heterotrimer and a membrane-targeting heterodimer or homodimer of SNX1 and/or SNX2 (ref. 13). Here we report the crystal structure of a VPS29-VPS35 subcomplex showing how the metallophosphoesterase-fold subunit VPS29 (refs 14, 15) acts as a scaffold for the carboxy-terminal half of VPS35. VPS35 forms a horseshoe-shaped, right-handed, alpha-helical solenoid, the concave face of which completely covers the metal-binding site of VPS29, whereas the convex face exposes a series of hydrophobic interhelical grooves. Electron microscopy shows that the intact VPS26-VPS29-VPS35 complex is a stick-shaped, flexible structure, approximately 21 nm long. A hybrid structural model derived from crystal structures, electron microscopy, interaction studies and bioinformatics shows that the alpha-solenoid fold extends the full length of VPS35, and that VPS26 is bound at the opposite end from VPS29. This extended structure presents multiple binding sites for the SNX complex and receptor cargo, and appears capable of flexing to conform to curved vesicular membranes.

  4. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

    PubMed

    Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

    2016-06-21

    The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood

  5. Recognition of Cognate Transfer RNA by the 30S Ribosomal Subunit

    SciTech Connect

    Ogle, James M.; Brodersen, Ditlev E.; Clemons, William M.; Tarry, Michael J.; Carter, Andrew P.; Ramakrishnan, V.

    2009-10-07

    Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or 'wobble,' position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.

  6. Structural Basis for Promoter ;#8722;10 Element Recognition by the Bacterial RNA Polymerase [sigma] Subunit

    SciTech Connect

    Feklistov, Andrey; Darst, Seth A.

    2011-12-15

    The key step in bacterial promoter opening is recognition of the -10 promoter element (T-{sub 12}A-{sub 11}T-{sub 10}A-{sub 9}A-{sub 8}T{sub -7} consensus sequence) by the RNA polymerase {alpha} subunit. We determined crystal structures of {alpha} domain 2 bound to single-stranded DNA bearing -10 element sequences. Extensive interactions occur between the protein and the DNA backbone of every -10 element nucleotide. Base-specific interactions occur primarily with A{sub -11} and T{sub -7}, which are flipped out of the single-stranded DNA base stack and buried deep in protein pockets. The structures, along with biochemical data, support a model where the recognition of the -10 element sequence drives initial promoter opening as the bases of the nontemplate strand are extruded from the DNA double-helix and captured by {alpha}. These results provide a detailed structural basis for the critical roles of A{sub -11} and T{sub -7} in promoter melting and reveal important insights into the initiation of transcription bubble formation.

  7. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    PubMed Central

    Beier, Anna; Krisp, Christoph; Wolters, Dirk A.

    2016-01-01

    ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. PMID:27329756

  8. Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica.

    PubMed

    Abdrakhmanova, Albina; Zickermann, Volker; Bostina, Mihnea; Radermacher, Michael; Schägger, Hermann; Kerscher, Stefan; Brandt, Ulrich

    2004-07-23

    Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.

  9. Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking.

    PubMed

    Tursun, Ablat; Zhu, Shaotong; Vik, Steven B

    2016-12-01

    Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

    PubMed

    Xie, Lili; Wartchow, Charles; Shia, Steven; Uehara, Kyoko; Steffek, Micah; Warne, Robert; Sutton, James; Muiru, Gladys T; Leonard, Vincent H J; Bussiere, Dirksen E; Ma, Xiaolei

    2016-01-01

    Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Recognition of overlapping nucleotides by AraC and the sigma subunit of RNA polymerase.

    PubMed

    Dhiman, A; Schleif, R

    2000-09-01

    The Escherichia coli promoter p(BAD), under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon. The binding site of AraC at p(BAD) overlaps the RNA polymerase -35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC. This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the -35 region or whether both AraC and sigma make important contacts with the DNA in the -35 region. If sigma does not contact DNA near the -35 region, p(BAD) activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC. We have examined this issue in the p(BAD) promoter and in a second promoter where the AraC binding site overlaps the -35 region by only 2 bases. In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the -35 region. Since sigma and AraC are thus closely positioned at p(BAD), it is possible that AraC and sigma contact one another during transcription initiation. DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.

  12. Subunit connectivity, assembly determinants, and architecture of the yeast exocyst complex

    PubMed Central

    Heider, Margaret R.; Gu, Mingyu; Duffy, Caroline M.; Mirza, Anne M.; Marcotte, Laura L.; Walls, Alexandra C.; Farrall, Nicholas; Hakhverdyan, Zhanna; Field, Mark C.; Rout, Michael P.; Frost, Adam; Munson, Mary

    2016-01-01

    The exocyst is a hetero-octameric complex proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst from Saccharomyces cerevisiae, and show that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin-induced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests the subunits are contiguous helical bundles packed together into a bundle of long rods. PMID:26656853

  13. A novel isoform of the human mitochondrial complex I subunit NDUFV3.

    PubMed

    Dibley, Marris G; Ryan, Michael T; Stroud, David A

    2017-01-01

    Human mitochondrial complex I is the first enzyme of the mitochondrial respiratory chain. Complex I is composed of 45 subunits, seven encoded by mitochondrial DNA, while the remainder are encoded by nuclear DNA. All nuclear-encoded subunits are thought to be expressed as a single isoform. Here we reveal subunit NDUFV3 to be present in both the canonical 10 kDa and a novel 50 kDa isoform, generated through alternative splicing. Both isoforms assemble into complex I and their levels vary in different tissues. While the 50 kDa isoform appears to be dominant in HEK293T cells, we find either isoform alone is sufficient for assembly of mature complex I. NDUFV3 represents the first known complex I subunit present in two functional isoforms. © 2016 Federation of European Biochemical Societies.

  14. Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components*

    PubMed Central

    Ghosh, Soma; Vassilev, Alex P.; Zhang, Junmei; Zhao, Yingming; DePamphilis, Melvin L.

    2011-01-01

    Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2–5) complex in the nucleus, an ORC(1–5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1–6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2–5) are transported independently to the nucleus where they can either assemble into ORC(1–6) or function individually. PMID:21555516

  15. The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

    PubMed Central

    Guo, Xiaoli; Mattera, Rafael; Ren, Xuefeng; Chen, Yu; Retamal, Claudio; González, Alfonso; Bonifacino, Juan S.

    2014-01-01

    SUMMARY An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the μ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous μ1A and the epithelial-specific μ1B. Previous studies led to the notion that μ1A and μ1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that μ1A and μ1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, μ1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a μ1B-dependent manner. We conclude that expression of distinct μ1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface. PMID:24229647

  16. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

    PubMed Central

    Ilca, Serban L.; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M.; Stuart, David I.; Huiskonen, Juha T.

    2015-01-01

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems. PMID:26534841

  17. Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit

    PubMed Central

    Tatebe, Hisashi; Murayama, Shinichi; Yonekura, Toshiya; Hatano, Tomoyuki; Richter, David; Furuya, Tomomi; Kataoka, Saori; Furuita, Kyoko; Kojima, Chojiro; Shiozaki, Kazuhiro

    2017-01-01

    The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT. DOI: http://dx.doi.org/10.7554/eLife.19594.001 PMID:28264193

  18. Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit.

    PubMed

    Tatebe, Hisashi; Murayama, Shinichi; Yonekura, Toshiya; Hatano, Tomoyuki; Richter, David; Furuya, Tomomi; Kataoka, Saori; Furuita, Kyoko; Kojima, Chojiro; Shiozaki, Kazuhiro

    2017-03-07

    The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT.

  19. Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

    PubMed Central

    Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

    2013-01-01

    Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

  20. Congenital deficiency of a 20-kDa subunit of mitochondrial complex I in fibroblasts.

    PubMed Central

    Slipetz, D M; Goodyer, P R; Rozen, R

    1991-01-01

    The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I. Images Figure 2 Figure 3 PMID:1903590

  1. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    PubMed

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility.

  2. Differential functions of WAVE regulatory complex subunits in the regulation of actin-driven processes.

    PubMed

    Litschko, Christof; Linkner, Joern; Brühmann, Stefan; Stradal, Theresia E B; Reinl, Tobias; Jänsch, Lothar; Rottner, Klemens; Faix, Jan

    2017-09-04

    The WAVE regulatory complex (WRC) links upstream Rho-family GTPase signaling to the activation of the ARP2/3 complex in different organisms. WRC-induced and ARP2/3 complex-mediated actin nucleation beneath the plasma membrane is critical for actin assembly in the leading edge to drive efficient cell migration. The WRC is a stable heteropentamer composed of SCAR/WAVE, Abi, Nap, Pir and the small polypeptide Brk1/Hspc300. Functional interference with individual subunits of the complex frequently results in diminished amounts of the remaining polypeptides of the WRC complex, implying the complex to act as molecular entity. However, Abi was also found to associate with mammalian N-WASP, formins, Eps8/SOS1 or VASP, indicating additional functions of individual WRC subunits in eukaryotic cells. To address this issue systematically, we inactivated all WRC subunits, either alone or in combination with VASP in Dictyostelium cells and quantified the protein content of the remaining subunits in respective WRC knockouts. The individual mutants displayed highly differential phenotypes concerning various parameters, including cell morphology, motility, cytokinesis or multicellular development, corroborating the view of additional roles for individual subunits, beyond their established function in WRC-mediated Arp2/3 complex activation. Finally, our data uncover the interaction of the actin polymerase VASP with WRC-embedded Abi to mediate VASP accumulation in cell protrusions, driving efficient cell migration. Copyright © 2017 Elsevier GmbH. All rights reserved.

  3. Deletion of the GluA1 AMPA receptor subunit impairs recency-dependent object recognition memory

    PubMed Central

    Sanderson, David J.; Hindley, Emma; Smeaton, Emily; Denny, Nick; Taylor, Amy; Barkus, Chris; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

    2011-01-01

    Deletion of the GluA1 AMPA receptor subunit impairs short-term spatial recognition memory. It has been suggested that short-term recognition depends upon memory caused by the recent presentation of a stimulus that is independent of contextual–retrieval processes. The aim of the present set of experiments was to test whether the role of GluA1 extends to nonspatial recognition memory. Wild-type and GluA1 knockout mice were tested on the standard object recognition task and a context-independent recognition task that required recency-dependent memory. In a first set of experiments it was found that GluA1 deletion failed to impair performance on either of the object recognition or recency-dependent tasks. However, GluA1 knockout mice displayed increased levels of exploration of the objects in both the sample and test phases compared to controls. In contrast, when the time that GluA1 knockout mice spent exploring the objects was yoked to control mice during the sample phase, it was found that GluA1 deletion now impaired performance on both the object recognition and the recency-dependent tasks. GluA1 deletion failed to impair performance on a context-dependent recognition task regardless of whether object exposure in knockout mice was yoked to controls or not. These results demonstrate that GluA1 is necessary for nonspatial as well as spatial recognition memory and plays an important role in recency-dependent memory processes. PMID:21378100

  4. The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization.

    PubMed

    Boesze-Battaglia, Kathleen; Walker, Lisa P; Zekavat, Ali; Dlakić, Mensur; Scuron, Monika Damek; Nygren, Patrik; Shenker, Bruce J

    2015-10-01

    Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.

  5. The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization

    PubMed Central

    Boesze-Battaglia, Kathleen; Walker, Lisa P.; Zekavat, Ali; Dlakić, Mensur; Scuron, Monika Damek; Nygren, Patrik

    2015-01-01

    Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells. PMID:26216427

  6. Oxo-anion recognition by mono- and bisurea pendant-arm macrocyclic complexes.

    PubMed

    Boiocchi, Massimo; Licchelli, Maurizio; Milani, Michele; Poggi, Antonio; Sacchi, Donatella

    2015-01-05

    The novel macrocyclic copper(II) complexes [2](2+) and [3](2+), carrying one or two (nitrophenyl)urea fragments appended to an azacyclam or diazacyclam framework, exploit the hydrogen-bond-forming abilities of the urea subunits, along with the metal-ligand interaction, in the recognition of anionic species. Equilibrium studies in acetonitrile performed on [2](2+) and [3](2+) show that (nitrophenyl)urea pendant arms strongly interact with anionic species such as carboxylates and phosphates, which display both coordinating tendencies toward copper(II) and good affinity toward urea subunits. Stability constants of the adducts are considerably higher than those determined for the interaction of the same anions with a "plain urea" reference compound, confirming the synergistic action of metallomacrocyclic and urea subunits. Complex [2](2+) forms 1:1 adducts with acetate, benzoate, hydrogendiphosphate, and dihydrogen phosphate, while complex [3](2+) interacts with the same anions according to both 1:1 and 1:2 stoichiometries, with the exception of hydrogendiphosphate, which forms only the 1:1 adduct with a distinctly high association constant (log K > 7). Spectrophotometric investigations suggest that oxoanionic species interact with the complexes according to a "bridged" mode, inducing the macrocyclic systems to adopt a scorpionate-like conformation, as confirmed by crystallographic studies on the [3](2+)/succinate adduct.

  7. Detailed analysis of the human mitochondrial contact site complex indicate a hierarchy of subunits.

    PubMed

    Ott, Christine; Dorsch, Eva; Fraunholz, Martin; Straub, Sebastian; Kozjak-Pavlovic, Vera

    2015-01-01

    Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components.

  8. Complex cell prototype representation for face recognition.

    PubMed

    Prssoa, L; Leitao, A P

    1999-01-01

    In this paper we propose a new face recognition system based on a biologically inspired filtering method. Our work differs from previous proposals in: 1) the multistage filtering method employed; 2) the pyramid structure used, and most importantly; 3) the prototype construction scheme to determine the models stored in memory. The method is much simpler than previous proposals and relatively inexpensive computationally, while attaining error rates as low as 5%, very close to the best reported results.

  9. Assembly of proteasome subunits into non-canonical complexes in vivo.

    PubMed

    Hammack, Lindsay J; Kusmierczyk, Andrew R

    2017-01-01

    Proteasomes exist in all domains of life. In general, they are comprised of a compartmentalized protease whose activity is modulated by one or more regulatory complexes with which it interacts. The quaternary structure of this compartmentalized protease, called the 20S proteasome, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and β subunits. In eukaryotes, assembly factors chaperone the α and β subunits during 20S biogenesis. Here we demonstrate that proteasome subunits can assemble into structures other than the canonical 20S proteasome in vivo. Specifically, the yeast α4 subunit forms high molecular weight complexes whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring-shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non-canonical complexes exist in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from Methanosarcina acetivorans▿ †

    PubMed Central

    Chen, Yi-Hsing; Lin, Yuyen; Yoshinaga, Aya; Chhotani, Benazir; Lorenzini, Jenna L.; Crofts, Alexander A.; Mei, Shou; Mackie, Roderick I.; Ishino, Yoshizumi; Cann, Isaac K. O.

    2009-01-01

    Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the Methanosarcina acetivorans clamp loader or replication factor C (RFC) homolog. M. acetivorans RFC (RFCMa), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal Escherichia coli clamp loader that exists in space as three γ-subunits (motor) flanked by the δ′ (stator) and the δ (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFCMa complex. PMID:19717601

  11. Structural and functional characterization of cargo-binding sites on the μ4-subunit of adaptor protein complex 4.

    PubMed

    Ross, Breyan H; Lin, Yimo; Corales, Esteban A; Burgos, Patricia V; Mardones, Gonzalo A

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  12. Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

    PubMed Central

    Ross, Breyan H.; Lin, Yimo; Corales, Esteban A.; Burgos, Patricia V.; Mardones, Gonzalo A.

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  13. Characterization of the origin recognition complex (ORC) from a higher plant, rice (Oryza sativa L.).

    PubMed

    Mori, Yoko; Yamamoto, Taichi; Sakaguchi, Norihiro; Ishibashi, Toyotaka; Furukawa, Tomoyuki; Kadota, Yasuhiro; Kuchitsu, Kazuyuki; Hashimoto, Junji; Kimura, Seisuke; Sakaguchi, Kengo

    2005-06-20

    The origin recognition complex (ORC) protein plays a critical role in DNA replication through binding to sites (origins) where replication commences. The protein is composed of six subunits (ORC1 to 6) in animals and yeasts. Our knowledge of the ORC protein in plants is, however, much less complete. We have performed cDNA cloning and characterization of ORC subunits in rice (Oryza sativa L. cv. Nipponbare) in order to facilitate study of plant DNA replication mechanisms. Our previous report provided a description of a gene, ORC1 (OsORC1), that encodes one of the protein subunits. The present report extends this initial analysis to include the genes that encode four other rice ORC subunits, OsORC2, 3, 4 and 5. Northern hybridization analyses demonstrated the presence of abundant transcripts for all OsORC subunits in shoot apical meristems (SAM) and cultured cells, but not in mature leaves. Interestingly, only OsORC5 showed high levels of expression in organs in which cell proliferation is not active, such as flag leaves, the ears and the non-tip roots. The pattern of expression of OsORC2 also differed from other OsORC subunits. When cell proliferation was temporarily halted for 6-10 days by removal of sucrose from the growth medium, expression of OsORC1, OsORC3, OsORC4 and OsORC5 was substantially reduced. However, the level of expression of OsORC2 remained constant. We suggest from these results that expression of OsORC1, 3, 4 and 5 are correlated with cell proliferation, but the expression of OsORC2 is not.

  14. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement.

    PubMed

    Setiaputra, Dheva T; Cheng, Derrick Th; Lu, Shan; Hansen, Jesse M; Dalwadi, Udit; Lam, Cindy Hy; To, Jeffrey L; Dong, Meng-Qiu; Yip, Calvin K

    2017-02-01

    Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.

  15. The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex

    PubMed Central

    Anwari, Khatira; Webb, Chaille T; Poggio, Sebastian; Perry, Andrew J; Belousoff, Matthew; Celik, Nermin; Ramm, Georg; Lovering, Andrew; Sockett, R Elizabeth; Smit, John; Jacobs-Wagner, Christine; Lithgow, Trevor

    2012-01-01

    The β-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, β-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli. PMID:22524202

  16. COG Complex Complexities: Detailed Characterization of a Complete Set of HEK293T Cells Lacking Individual COG Subunits

    PubMed Central

    Bailey Blackburn, Jessica; Pokrovskaya, Irina; Fisher, Peter; Ungar, Daniel; Lupashin, Vladimir V.

    2016-01-01

    The Conserved Oligomeric Golgi complex is an evolutionarily conserved multisubunit tethering complex (MTC) that is crucial for intracellular membrane trafficking and Golgi homeostasis. The COG complex interacts with core vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is still an enigma. Previous studies of COG complex were limited to the use of CDGII (Congenital disorders of glycosylation type II)-COG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches. In this study we have used the CRISPR approach to generate HEK293T knock-out (KO) cell lines missing individual COG subunits. These cell lines were characterized for glycosylation and trafficking defects, cell proliferation rates, stability of COG subunits, localization of Golgi markers, changes in Golgi structure, and N-glycan profiling. We found that all KO cell lines were uniformly deficient in cis/medial-Golgi glycosylation and each had nearly abolished binding of Cholera toxin. In addition, all cell lines showed defects in Golgi morphology, retrograde trafficking and sorting, sialylation and fucosylation, but severities varied according to the affected subunit. Lobe A and Cog6 subunit KOs displayed a more severely distorted Golgi structure, while Cog2, 3, 4, 5, and 7 knock outs had the most hypo glycosylated form of Lamp2. These results led us to conclude that every subunit is essential for COG complex function in Golgi trafficking, though to varying extents. We believe that this study and further analyses of these cells will help further elucidate the roles of individual COG subunits and bring a greater understanding to the class of MTCs as a whole. PMID:27066481

  17. Thermodynamic computational approach to capture molecular recognition in the binding of different inhibitors to the DNA gyrase B subunit from Escherichia coli.

    PubMed

    Saíz-Urra, Liane; Pérez, Miguel Ángel Cabrera; Froeyen, Matheus

    2013-08-01

    DNA gyrase subunit B, that catalyzes the hydrolysis of ATP, is an attractive target for the development of antibacterial drugs. This work is intended to rationalize molecular recognition at DNA gyrase B enzyme - inhibitor binding interface through the evaluation of different scoring functions in finding the correct pose and scoring properly 50 Escherichia coli DNA Gyrase B inhibitors belonging to five different classes. Improving the binding free energy calculation accuracy is further attempted by using rescoring schemes after short molecular dynamic simulations of the obtained docked complexes. These data are then compared with the corresponding experimental enzyme activity data. The results are analyzed from a structural point of view emphasizing the strengths and limitations of the techniques applied in the study.

  18. Redefining the roles of mitochondrial DNA-encoded subunits in respiratory Complex I assembly

    PubMed Central

    Vartak, Rasika; Deng, Janice; Fang, Hezhi; Bai, Yidong

    2015-01-01

    Respiratory Complex I deficiency is implicated in numerous degenerative and metabolic diseases. In particular, mutations in several mitochondrial DNA (mtDNA)-encoded Complex I subunits including ND4, ND5 and ND6 have been identified in several neurological diseases. We previously demonstrated that these subunits played essential roles in Complex I assembly which in turn affected mitochondrial function. Here, we carried out a comprehensive study of the Complex I assembly pathway. We identified a new Complex I intermediate containing both membrane and matrix arms at an early assembly stage. We find that lack of the ND6 subunit does not hinder membrane arm formation; instead it recruits ND1 and ND5 enter the intermediate. While ND4 is important for the formation of the newly identified intermediate, the addition of ND5 stabilizes the complex and is required for the critical transition from Complex I to supercomplexes assembly. As a result, the Complex I assembly pathway has been redefined in this study. PMID:25887158

  19. Spatial subunit distribution and in vitro functions of the novel trimeric PCNA complex from Sulfolobus tokodaii

    SciTech Connect

    Lu Shuhong; Li Zhuo; Wang Zhiyu; Ma Xiaoqing; Sheng Duohong; Ni Jinfeng; Shen Yulong

    2008-11-14

    The relationships among three PCNA (proliferating cell nuclear antigen) subunits in the hyperthermophilic archaeon Sulfolobus tokodaii (StoPCNAs) were analyzed and the effects of two PCNA complexes on the activities of the DNA helicase Hjm, DNA Ligase I, and Holliday junction specific endonuclease Hjc were tested. There was no strong self-interaction of each StoPCNA. StoPCNA1 and StoPCNA3 interacted with each other, so did StoPCNA2 and StoPCNA3, but no interaction between StoPCNA1 and StoPCNA2 was observed. Two trimeric complexes (designed StoPCNA123 and StoPCNA323) were formed in vitro and it was determined that StoPCNA323 was composed of one StoPCNA2 and two StoPCNA3 subunits, with StoPCNA2 bridging the two StoPCNA3 subunits. Both complexes inhibited the unwinding activity of Hjm and the ligation activity of DNA Ligase I. In contrast, both stimulated the Holliday junction cleavage activity of Hjc. Our results provide further evidence that in crenarchaea, the PCNAs exhibit diversity in subunit interaction and complex formation.

  20. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  1. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  2. Interactions among rice ORC subunits.

    PubMed

    Tan, Deyong; Lv, Qundan; Chen, Xinai; Shi, Jianghua; Ren, Meiyan; Wu, Ping; Mao, Chuanzao

    2013-08-01

    The origin recognition complex (ORC) is composed of six subunits and plays an important role in DNA replication in all eukaryotes. The ORC subunits OsORC6 as well as the other five ORC subunits in rice were experimentally isolated and sequenced. It indicated that there also exist six ORC subunits in rice. Results of RT-PCR indicated that expression of all the rice ORC genes are no significant difference under 26°C and 34°C. Yeast two hybridization indicated that OsORC2, -3, -5 interact with each other. OsORC5 can then bind OsORC4 to form the OsORC2, -3,-4,-5 core complex. It suggested that the basic interactions have been conserved through evolution. No binding of OsORC1 and OsORC6 with the other subunits were observed. A model of ORC complex in rice is proposed.

  3. Identification and evolutionary analysis of tissue-specific isoforms of mitochondrial complex I subunit NDUFV3.

    PubMed

    Guerrero-Castillo, Sergio; Cabrera-Orefice, Alfredo; Huynen, Martijn A; Arnold, Susanne

    2017-03-01

    Mitochondrial complex I is the largest respiratory chain complex. Despite the enormous progress made studying its structure and function in recent years, potential regulatory roles of its accessory subunits remained largely unresolved. Complex I gene NDUFV3, which occurs in metazoa, contains an extra exon that is only present in vertebrates and thereby evolutionary even younger than the rest of the gene. Alternative splicing of this extra exon gives rise to a short NDUFV3-S and a long NDUFV3-L protein isoform. Complexome profiling revealed that the two NDUFV3 isoforms are constituents of the multi-subunit complex I. Further mass spectrometric analyses of complex I from different murine and bovine tissues showed a tissue-specific expression pattern of NDUFV3-S and NDUFV3-L. Hence, NDUFV3-S was identified as the only isoform in heart and skeletal muscle, whereas in liver, brain, and lung NDUFV3-L was expressed as the dominant isoform, together with NDUFV3-S present in all tissues analyzed. Thus, we identified NDUFV3 as the first out of 30 accessory subunits of complex I present in vertebrate- and tissue-specific isoforms. Interestingly, the tissue-specific expression pattern of NDUFV3-S and NDUFV3-L isoforms was paralleled by changes in kinetic parameters, especially the substrate affinity of complex I. This may indicate a regulatory role of the NDUFV3 isoforms in different vertebrate tissues.

  4. Complexity reduction with recognition rate maintained for online handwritten Japanese text recognition

    NASA Astrophysics Data System (ADS)

    Gao, Jinfeng; Zhu, Bilan; Nakagawa, Masaki

    2012-01-01

    The paper presents complexity reduction of an on-line handwritten Japanese text recognition system by selecting an optimal off-line recognizer in combination with an on-line recognizer, geometric context evaluation and linguistic context evaluation. The result is that a surprisingly small off-line recognizer, which alone is weak, produces nearly the best recognition rate in combination with other evaluation factors in remarkably small space and time complexity. Generally speaking, lower dimensions with less principle components produce a smaller set of prototypes, which reduce memory-cost and time-cost. It degrades the recognition rate, however, so that we need to compromise them. In an evaluation function with the above-mentioned multiple factors combined, the configuration of only 50 dimensions with as little as 5 principle components for the off-line recognizer keeps almost the best accuracy 97.87% (the best accuracy 97.92%) for text recognition while it suppresses the total memory-cost from 99.4 MB down to 32 MB and the average time-cost of character recognition for text recognition from 0.1621 ms to 0.1191 ms compared with the traditional offline recognizer with 160 dimensions and 50 principle components.

  5. Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

    PubMed Central

    Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.

    2014-01-01

    ABSTRACT The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58-amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex. PMID:25352624

  6. Recognition and Management of Medical Complexity.

    PubMed

    Kuo, Dennis Z; Houtrow, Amy J

    2016-12-01

    Children with medical complexity have extensive needs for health services, experience functional limitations, and are high resource utilizers. Addressing the needs of this population to achieve high-value health care requires optimizing care within the medical home and medical neighborhood. Opportunities exist for health care providers, payers, and policy makers to develop strategies to enhance care delivery and to decrease costs. Important outcomes include decreasing unplanned hospital admissions, decreasing emergency department use, ensuring access to health services, limiting out-of-pocket expenses for families, and improving patient and family experiences, quality of life, and satisfaction with care. This report describes the population of children with medical complexity and provides strategies to optimize medical and health outcomes. Copyright © 2016 by the American Academy of Pediatrics.

  7. Structural Characterization of Tip20p and Dsl1p, Subunits of the Dsl1p Vesicle Tethering Complex

    SciTech Connect

    Tripathi, A.; Ren, Y; Jeffrey, P; Hughson, F

    2009-01-01

    Multisubunit tethering complexes are essential for intracellular trafficking and have been proposed to mediate the initial interaction between vesicles and the membranes with which they fuse. Here we report initial structural characterization of the Dsl1p complex, whose three subunits are essential for trafficking from the Golgi apparatus to the endoplasmic reticulum (ER). Crystal structures reveal that two of the three subunits, Tip20p and Dsl1p, resemble known subunits of the exocyst complex, establishing a structural connection among several multisubunit tethering complexes and implying that many of their subunits are derived from a common progenitor. We show, moreover, that Tip20p and Dsl1p interact directly via N-terminal alpha-helices. Finally, we establish that different Dsl1p complex subunits bind independently to different ER SNARE proteins. Our results map out two alternative protein-interaction networks capable of tethering COPI-coated vesicles, via the Dsl1p complex, to ER membranes.

  8. Research on recognition methods of aphid objects in complex backgrounds

    NASA Astrophysics Data System (ADS)

    Zhao, Hui-Yan; Zhang, Ji-Hong

    2009-07-01

    In order to improve the recognition accuracy among the kinds of aphids in the complex backgrounds, the recognition method among kinds of aphids based on Dual-Tree Complex Wavelet Transform (DT-CWT) and Support Vector Machine (Libsvm) is proposed. Firstly the image is pretreated; secondly the aphid images' texture feature of three crops are extracted by DT-CWT in order to get the training parameters of training model; finally the training model could recognize aphids among the three kinds of crops. By contrasting to Gabor wavelet transform and the traditional extracting texture's methods based on Gray-Level Co-Occurrence Matrix (GLCM), the experiment result shows that the method has a certain practicality and feasibility and provides basic for aphids' recognition between the identification among same kind aphid.

  9. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes

    PubMed Central

    Dawe, G. Brent; Musgaard, Maria; Aurousseau, Mark R.P.; Nayeem, Naushaba; Green, Tim; Biggin, Philip C.; Bowie, Derek

    2016-01-01

    Summary Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits. PMID:26924438

  10. A scaffold of accessory subunits links the peripheral arm and the distal proton pumping module of mitochondrial complex I

    PubMed Central

    ANGERER, Heike; ZWICKER, Klaus; WUMAIER, Zibiernisha; SOKOLOVA, Lucie; HEIDE, Heinrich; STEGER, Mirco; KAISER, Silke; NÜBEL, Esther; BRUTSCHY, Bernhard; RADERMACHER, Michael; BRANDT, Ulrich; ZICKERMANN, Volker

    2011-01-01

    Synopsis Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I. PMID:21545356

  11. Isolation of Thylakoid Membrane Complexes from Rice by a New Double-Strips BN/SDS-PAGE and Bioinformatics Prediction of Stromal Ridge Subunits Interaction

    PubMed Central

    Yu, Jianlan; Guo, Lin; Ding, Yi

    2011-01-01

    Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new “Double-Strips BN/SDS-PAGE” method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy. PMID:21637806

  12. Multiclass pattern recognition using adaptive correlation filters with complex constraints

    NASA Astrophysics Data System (ADS)

    Diaz-Ramirez, Victor H.; Campos-Trujillo, Oliver G.; Kober, Vitaly; Aguilar-Gonzalez, Pablo M.

    2012-03-01

    An efficient method for reliable multiclass pattern recognition using a bank of adaptive correlation filters is proposed. The method can recognize and classify multiple targets from an input scene by using both the intensity and phase distributions of the output complex correlation plane. The adaptive filters are synthesized with the help of an iterative algorithm based on synthetic discriminant functions with complex constraints. The algorithm optimizes the discrimination capability of the adaptive filters and determines the minimum number of filters in a bank to guarantee a desired classification efficiency. As a result, the computational complexity of the proposed system is low. Computer simulation results obtained with the proposed approach in cluttered and noisy scenes are discussed and compared with those obtained through existing methods in terms of recognition performance, classification efficiency, and computational complexity.

  13. Skills Recognition and Validation--Complexity and Tensions

    ERIC Educational Resources Information Center

    Cavaco, Carmen

    2009-01-01

    This article seeks to identify and examine the reasons for the complexity and tensions underlying the skills recognition, accreditation and certification scheme (SRAC) that has been in place in Portugal since 2001. Empirical data were collected through semi-directive interviews with staff in three Centros Novas Oportunidades [CNOs] [New…

  14. Distinct contributions of MSL complex subunits to the transcriptional enhancement responsible for dosage compensation in Drosophila.

    PubMed

    Dunlap, David; Yokoyama, Ruth; Ling, Huiping; Sun, He-Ying; McGill, Kerry; Cugusi, Simona; Lucchesi, John C

    2012-12-01

    The regulatory mechanism of dosage compensation is the paramount example of epigenetic regulation at the chromosomal level. In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males. We have investigated the function of various subunits of the complex in mediating dosage compensation. Our results confirm that the highly enriched specific acetylation of histone H4 at lysine 16 of compensated genes by the histone acetyl transferase subunit MOF induces a more disorganized state of their chromatin. We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification. Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

  15. Identification and linkage of the proteasome activator complex PA28 subunit genes in zebrafish.

    PubMed

    Murray, B W; Sültmann, H; Klein, J

    2000-06-01

    PA28 is an activator of the latent 20S proteasome, a large multisubunit complex involved in intracellular proteolysis. Two forms of hexameric PA28 have been identified, PA28-(alphabeta)3 and PA28-(gamma)6, of which the former is of immunological importance. Both the PA28-alpha and PA28-beta subunits are inducible by interferon-gamma (IFN-gamma) and the PA28-(alphabeta)3 complex enhances the ability of the 20S proteasome to produce peptides suited for binding to major histocompatibility complex (Mhc) class I molecules. To identify the homologues of the PA28 subunits in zebrafish we screened a cDNA library and obtained full-length cDNA sequences of the genes PSME1, PSME2 and PSME3 coding for the PA28-alpha, PA28-beta and PA28-gamma subunits, respectively. Phylogenetic analysis indicates the existence of the ancestors of all three genes prior to the divergence of tetrapods and bony fishes. The IFN-gamma-inducible subunits, PA28-alpha and PA28-beta, evolve faster than the presumably older PA28-gamma subunit. Using zebrafish radiation hybrid panels, the genes PSME2 and PSME3 were mapped to linkage group 12 and shown to be separated by a distance of less than 2.4 cM. This observation suggests that an intrachromosomal duplication event created the precursor of the IFN-gamma-inducible genes from a PA28-gamma-like ancestor prior to their recruitment into the Mhc class I peptide presentation pathway.

  16. Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

    PubMed

    Bridges, Hannah R; Mohammed, Khairunnisa; Harbour, Michael E; Hirst, Judy

    2017-03-01

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.

  17. Emotional states recognition, implementing a low computational complexity strategy.

    PubMed

    Aguiñaga, Adrian Rodriguez; Ramirez, Miguel Angel Lopez

    2016-09-18

    This article describes a methodology to recognize emotional states through an electroencephalography signals analysis, developed with the premise of reducing the computational burden that is associated with it, implementing a strategy that reduces the amount of data that must be processed by establishing a relationship between electrodes and Brodmann regions, so as to discard electrodes that do not provide relevant information to the identification process. Also some design suggestions to carry out a pattern recognition process by low computational complexity neural networks and support vector machines are presented, which obtain up to a 90.2% mean recognition rate.

  18. Crystal structure of Sec10, a subunit of the exocyst complex

    PubMed Central

    Chen, Jianxing; Yamagata, Atsushi; Kubota, Keiko; Sato, Yusuke; Goto-Ito, Sakurako; Fukai, Shuya

    2017-01-01

    The exocyst complex is a heterooctameric protein complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. This complex plays an essential role in trafficking secretory vesicles to the plasma membrane through its interaction with phosphatidylinositol 4,5-bisphosphate and small GTPases. To date, the near-full-length structural information of each subunit has been limited to Exo70, although the C-terminal half structures of Sec6, Sec15 and Exo84 and the structures of the small GTPase-binding domains of Sec3, Sec5 and Exo84 have been reported. Here, we report the crystal structure of the near-full-length zebrafish Sec10 (zSec10) at 2.73 Å resolution. The structure of zSec10 consists of tandem antiparallel helix bundles that form a straight rod, like helical core regions of other exocyst subunits. This structure provides the first atomic details of Sec10, which may be useful for future functional and structural studies of this subunit and the exocyst complex. PMID:28098232

  19. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions

    PubMed Central

    Wiedenheft, Blake; van Duijn, Esther; Bultema, Jelle B.; Waghmare, Sakharam P.; Zhou, Kaihong; Barendregt, Arjan; Westphal, Wiebke; Heck, Albert J. R.; Boekema, Egbert J.; Dickman, Mark J.; Doudna, Jennifer A.

    2011-01-01

    Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1–4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a “seed sequence” at the 5′ end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems. PMID:21536913

  20. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions.

    PubMed

    Wiedenheft, Blake; van Duijn, Esther; Bultema, Jelle B; Bultema, Jelle; Waghmare, Sakharam P; Waghmare, Sakharam; Zhou, Kaihong; Barendregt, Arjan; Westphal, Wiebke; Heck, Albert J R; Heck, Albert; Boekema, Egbert J; Boekema, Egbert; Dickman, Mark J; Dickman, Mark; Doudna, Jennifer A

    2011-06-21

    Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.

  1. Core promoter recognition complex changes accompany liver development

    PubMed Central

    D’Alessio, Joseph A.; Ng, Raymond; Willenbring, Holger; Tjian, Robert

    2011-01-01

    Recent studies of several key developmental transitions have brought into question the long held view of the basal transcriptional apparatus as ubiquitous and invariant. In an effort to better understand the role of core promoter recognition and coactivator complex switching in cellular differentiation, we have examined changes in transcription factor IID (TFIID) and cofactor required for Sp1 activation/Mediator during mouse liver development. Here we show that the differentiation of fetal liver progenitors to adult hepatocytes involves a wholesale depletion of canonical cofactor required for Sp1 activation/Mediator and TFIID complexes at both the RNA and protein level, and that this alteration likely involves silencing of transcription factor promoters as well as protein degradation. It will be intriguing for future studies to determine if a novel and as yet unknown core promoter recognition complex takes the place of TFIID in adult hepatocytes and to uncover the mechanisms that down-regulate TFIID during this critical developmental transition. PMID:21368148

  2. PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation

    PubMed Central

    Yang, Yan; Li, Wencheng; Hoque, Mainul; Hou, Liming; Shen, Steven; Tian, Bin; Dynlacht, Brian D.

    2016-01-01

    The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and RNA polymerase II (PolII) elongation. Here, we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, we identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3’ ends of transcripts. Moreover, we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation, although there was little impact of Paf1C depletion on other histone modifications, including tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site (TSS)-proximal pA sites and regulating pA site choice in 3’UTRs. PMID:26765774

  3. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.

    PubMed

    Carroll, Joe; Shannon, Richard J; Fearnley, Ian M; Walker, John E; Hirst, Judy

    2002-12-27

    Mitochondrial NADH:ubiquinone oxidoreductase (complex I) from bovine heart is a complicated multisubunit, membrane-bound assembly. Seven subunits are encoded by mitochondrial DNA, and the sequences of 36 nuclear encoded subunits have been described. The subunits of complex I and two subcomplexes (Ialpha and Ibeta) were resolved on one- and two-dimensional gels and by reverse-phase high performance liquid chromatography. Mass spectrometric analysis revealed two previously unknown subunits in complex I, named B14.7 and ESSS, one in each subcomplex. Coding sequences for each protein were identified in data bases and were confirmed by cDNA cloning and sequencing. Subunit B14.7 has an acetylated N terminus, no presequence, and contains four potential transmembrane helices. It is homologous to subunit 21.3b from complex I in Neurospora crassa and is related to Tim17, Tim22, and Tim23, which are involved in protein translocation across the inner membrane. Subunit ESSS has a cleaved mitochondrial import sequence and one potential transmembrane helix. A total of 45 different subunits of bovine complex I have now been characterized.

  4. RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking.

    PubMed

    Lin, Xiaosi; Yang, Ting; Wang, Shicong; Wang, Zhen; Yun, Ye; Sun, Lixiang; Zhou, Yunhe; Xu, Xiaohui; Akazawa, Chihiro; Hong, Wanjin; Wang, Tuanlao

    2014-12-02

    The HOPS complex serves as a tethering complex with GEF activity for Ypt7p in yeast to regulate late endosomal membrane maturation. While the role of HOPS complex is well established in yeast cells, its functional and mechanistic aspects in mammalian cells are less well defined. In this study, we report that RILP, a downstream effector of Rab7, interacts with HOPS complex and recruits HOPS subunits to the late endosomal compartment. Structurally, the amino-terminal portion of RILP interacts with HOPS complex. Unexpectedly, this interaction is independent of Rab7. VPS41 subunit of HOPS complex was defined to be the major partner for interacting with RILP. The carboxyl-terminal region of VPS41 was mapped to be responsible for the interaction. Functionally, either depletion of VPS41 by shRNA or overexpression of VPS41 C-terminal half retarded EGF-induced degradation of EGFR. These results suggest that interaction of RILP with HOPS complex via VPS41 plays a role in endocytic trafficking of EGFR.

  5. SMC proteins constitute two subunits of the mammalian recombination complex RC-1.

    PubMed Central

    Jessberger, R; Riwar, B; Baechtold, H; Akhmedov, A T

    1996-01-01

    Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC-1. N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities. Images PMID:8670910

  6. Elg1, the major subunit of an alternative RFC complex, interacts with SUMO-processing proteins.

    PubMed

    Parnas, Oren; Amishay, Rona; Liefshitz, Batia; Zipin-Roitman, Adi; Kupiec, Martin

    2011-09-01

    PCNA is a homotrimeric ring with important roles in DNA replication and repair. PCNA is loaded and unloaded by the RFC complex, which is composed of five subunits (Rfc1-5). Three additional complexes that share with RFC the small subunits (Rfc2-5) and contain alternative large subunits were found in yeast and other eukaryotes. We have recently reported that one of these, the Elg1-RFC complex, interacts with SUMOylated PCNA and may play a role in its unloading during DNA repair. Here we report that a yeast-two-hybrid screen with the N terminus of Elg1(which interacts with SUMOylated PCNA) uncovered interactions with proteins that belong to the SUMO pathway, including Slx5 and Slx8, which form an E3 ubiquitin ligase that ubiquitinates SUMOylated proteins. Mutations in SLX5 result in a genomic instability phenotype similar to that of elg1 mutants. The physical interaction between the N terminus of Elg1 and Slx5 is mediated by poly-SUMO chains but not by PCNA modifications, and requires Siz2, but not Siz1, activity. Thus our results highlight the many important roles played by Elg1, some of which are PCNA-dependent and some PCNA-independent.

  7. The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes.

    PubMed Central

    Garriga, J; Mayol, X; Graña, X

    1996-01-01

    PITALRE is a human protein kinase identified by means of its partial sequence identity to the cell division cycle regulatory kinase CDC2. Immunopurified PITALRE protein complexes exhibit an in vitro kinase activity that phosphorylates the retinoblastoma protein, suggesting that PITALRE catalyses this phosphorylation reaction. However, the presence of other kinases in the immunopurified complex could not be ruled out. In the present work, an inactive mutant of the PITALRE kinase has been used to demonstrate that PITALRE is the catalytic subunit responsible for the PITALRE-complex-associated kinase activity, Ectopic overexpression of PITALRE did not increase the total PITALRE kinase activity in the cell, suggesting that PITALRE is regulated by limiting cellular factor(s). Characterization of the PITALRE-containing protein complexes indicated that most of the cellular PITALRE protein exists as a subunit in at least two different active multimeric complexes. Although monomeric PITALRE is also active in vitro, PITALRE present in multimeric complexes exhibits several-fold higher activity than monomeric PITALRE. In addition, overexpression of PITALRE demonstrated the existence of two new associated proteins of approx. 48 and 98 kDa. Altogether these results suggest that, in contrast to the situation with cyclin-dependent kinases, monomeric PITALRE is active, and that association with other proteins modulates its activity and/or its ability to recognize substrates in vivo. PMID:8870681

  8. A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I.

    PubMed

    Kashani-Poor, N; Zwicker, K; Kerscher, S; Brandt, U

    2001-06-29

    We have analyzed a series of eleven mutations in the 49-kDa protein of mitochondrial complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica to identify functionally important domains in this central subunit. The mutations were selected based on sequence homology with the large subunit of [NiFe] hydrogenases. None of the mutations affected assembly of complex I, all decreased or abolished ubiquinone reductase activity. Several mutants exhibited decreased sensitivities toward ubiquinone-analogous inhibitors. Unexpectedly, seven mutations affected the properties of iron-sulfur cluster N2, a prosthetic group not located in the 49-kDa subunit. In three of these mutants cluster N2 was not detectable by electron-paramagnetic resonance spectroscopy. The fact that the small subunit of hydrogenase is homologous to the PSST subunit of complex I proposed to host cluster N2 offers a straightforward explanation for the observed, unforeseen effects on this iron-sulfur cluster. We propose that the fold around the hydrogen reactive site of [NiFe] hydrogenase is conserved in the 49-kDa subunit of complex I and has become part of the inhibitor and ubiquinone binding region. We discuss that the fourth ligand of iron-sulfur cluster N2 missing in the PSST subunit may be provided by the 49-kDa subunit.

  9. Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture

    PubMed Central

    Tiengwe, Calvin; Marcello, Lucio; Farr, Helen; Gadelha, Catarina; Burchmore, Richard; Barry, J. David; Bell, Stephen D.; McCulloch, Richard

    2012-01-01

    DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing. PMID:22412905

  10. Face recognition using dual-tree complex wavelet features.

    PubMed

    Liu, Chao-Chun; Dai, Dao-Qing

    2009-11-01

    We propose a novel facial representation based on the dual-tree complex wavelet transform for face recognition. It is effective and efficient to represent the geometrical structures in facial image with low redundancy. Moreover, we experimentally verify that the proposed method is more powerful to extract facial features robust against the variations of shift and illumination than the discrete wavelet transform and Gabor wavelet transform.

  11. The viewpoint complexity of an object-recognition task.

    PubMed

    Tjan, B S; Legge, G E

    1998-08-01

    There is an ongoing debate about the nature of perceptual representation in human object recognition. Resolution of this debate has been hampered by the lack of a metric for assessing the representational requirements of a recognition task. To recognize a member of a given set of 3-D objects, how much detail must the objects' representations contain in order to achieve a specific accuracy criterion? From the performance of an ideal observer, we derived a quantity called the view complexity (VX) to measure the required granularity of representation. VX is an intrinsic property of the object-recognition task, taking into account both the object ensemble and the type of decision required of an observer. It does not depend on the visual representation or processing used by the observer. VX can be interpreted as the number of randomly selected 2-D images needed to represent the decision boundaries in the image space of a 3-D object-recognition task. A low VX means the task is inherently more viewpoint invariant and a high VX means it is inherently more viewpoint dependent. By measuring the VX of recognition tasks with different object sets, we show that the current confusion about the nature of human perceptual representation is partly due to a failure in distinguishing between human visual processing and the properties of a task and its stimuli. We find general correspondence between the VX of a recognition task and the published human data on viewpoint dependence. Exceptions in this relationship motivated us to propose the view-rate hypothesis: human visual performance is limited by the equivalent number of 2-D image views that can be processed per unit time.

  12. Mitochondrial Complex V α Subunit Is Critical for Candida albicans Pathogenicity through Modulating Multiple Virulence Properties

    PubMed Central

    Li, Shui-Xiu; Song, Yan-Jun; Zhang, Yi-Shan; Wu, Hao-Tian; Guo, Hui; Zhu, Kun-Ju; Li, Dong-Mei; Zhang, Hong

    2017-01-01

    The α subunit (ATP1) is a vital component of mitochondrial complex V which counts for the majority of cellular ATP production in a living organism. Nevertheless, how the α subunit influences other cellular processes such as pathogenicity in Candida albicans remains poorly understood. To address this question, ATP1 mutant (atp1Δ/Δ) and the gene-reconstituted strain (atp1Δ/ATP1) have been constructed in this study and their pathogenicity-related traits are compared to those of wild type (WT). In a murine model of disseminated candidiasis, atp1Δ/Δ infected mice have a significantly higher survival rate and experience a lower fungal burden in tissues. In in vitro studies atp1Δ/Δ lose a capability to damage or destroy macrophages and endothelial cells. Furthermore, atp1Δ/Δ is not able to grow under either glucose-denial conditions or high H2O2 conditions, both of which are associated with the potency of the macrophages to kill C. albicans. Defects in filamentation and biofilm formation may impair the ability of atp1Δ/Δ to penetrate host cells and establish robust colonies in the host tissues. In concert with these pathogenic features, intracellular ATP levels of atp1Δ/Δ can drop to 1/3 of WT level. These results indicate that the α subunit of Complex V play important roles in C. albicans pathogenicity. PMID:28280492

  13. Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

    SciTech Connect

    Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming; Hale, Caryn R.; Terns, Rebecca M.; Terns, Michael P.; Li, Hong

    2012-08-10

    Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

  14. Accessory subunit NUYM (NDUFS4) is required for stability of the electron input module and activity of mitochondrial complex I.

    PubMed

    Kahlhöfer, Flora; Kmita, Katarzyna; Wittig, Ilka; Zwicker, Klaus; Zickermann, Volker

    2017-02-01

    Mitochondrial complex I is an intricate 1MDa membrane protein complex with a central role in aerobic energy metabolism. The minimal form of complex I consists of fourteen central subunits that are conserved from bacteria to man. In addition, eukaryotic complex I comprises some 30 accessory subunits of largely unknown function. The gene for the accessory NDUFS4 subunit of human complex I is a hot spot for fatal pathogenic mutations in humans. We have deleted the gene for the orthologous NUYM subunit in the aerobic yeast Yarrowia lipolytica, an established model system to study eukaryotic complex I and complex I linked diseases. We observed assembly of complex I which lacked only subunit NUYM and retained weak interaction with assembly factor N7BML (human NDUFAF2). Absence of NUYM caused distortion of iron sulfur clusters of the electron input domain leading to decreased complex I activity and increased release of reactive oxygen species. We conclude that NUYM has an important stabilizing function for the electron input module of complex I and is essential for proper complex I function.

  15. Deacetylase inhibitors dissociate the histone-targeting ING2 subunit from the Sin3 complex

    PubMed Central

    Smith, Karen T.; Martin-Brown, Skylar A.; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

    2010-01-01

    Summary Histone deacetylase (HDAC) inhibitors are in clinical development for several diseases, including cancers and neurodegenerative disorders. HDACs1 and 2 are among the targets of these inhibitors and are part of multisubunit protein complexes. HDAC inhibitors (HDACi) block the activity of HDACs by chelating a zinc molecule in their catalytic sites. It is not known if the inhibitors have any additional functional effects on the multisubunit HDAC complexes. Here, we find that suberoylanilide hydroxamic acid (SAHA), the recently FDA approved HDACi, causes the dissociation of the PHD-finger containing ING2 subunit from the Sin3 deacetylase complex. Loss of ING2 disrupts the in vivo binding of the Sin3 complex to the p21 promoter, an important target gene for cell growth inhibition by SAHA. Our findings reveal a new molecular mechanism by which HDAC inhibitors disrupt deacetylase function. PMID:20142042

  16. The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

    SciTech Connect

    Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui; Wong, Sui-Lam; Ng, Kenneth K. S.

    2013-05-01

    The structure of the SBP-Tag–streptavidin complex reveals a novel mode of peptide recognition in which a single peptide binds simultaneously to biotin-binding pockets from adjacent subunits of streptavidin. The molecular details of peptide recognition suggest how the SBP-Tag can be further modified to become an even more useful tag for a wider range of biotechnological applications. The 38-residue SBP-Tag binds to streptavidin more tightly (K{sub d} ≃ 2.5–4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12–14) and a C-terminal HPQ sequence (residues 31–33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17–28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1–10 and 35–38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11–34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

  17. Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I

    PubMed Central

    Kmita, Katarzyna; Wirth, Christophe; Warnau, Judith; Guerrero-Castillo, Sergio; Hunte, Carola; Hummer, Gerhard; Kaila, Ville R. I.; Zwicker, Klaus; Brandt, Ulrich; Zickermann, Volker

    2015-01-01

    Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4. PMID:25902503

  18. Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation

    PubMed Central

    Marques, Catarina A.; Tiengwe, Calvin; Lemgruber, Leandro; Damasceno, Jeziel D.; Scott, Alan; Paape, Daniel; Marcello, Lucio; McCulloch, Richard

    2016-01-01

    Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying. PMID:26951375

  19. Pattern recognition tool based on complex network-based approach

    NASA Astrophysics Data System (ADS)

    Casanova, Dalcimar; Backes, André Ricardo; Martinez Bruno, Odemir

    2013-02-01

    This work proposed a generalization of the method proposed by the authors: 'A complex network-based approach for boundary shape analysis'. Instead of modelling a contour into a graph and use complex networks rules to characterize it, here, we generalize the technique. This way, the work proposes a mathematical tool for characterization signals, curves and set of points. To evaluate the pattern description power of the proposal, an experiment of plat identification based on leaf veins image are conducted. Leaf vein is a taxon characteristic used to plant identification proposes, and one of its characteristics is that these structures are complex, and difficult to be represented as a signal or curves and this way to be analyzed in a classical pattern recognition approach. Here, we model the veins as a set of points and model as graphs. As features, we use the degree and joint degree measurements in a dynamic evolution. The results demonstrates that the technique has a good power of discrimination and can be used for plant identification, as well as other complex pattern recognition tasks.

  20. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse

    PubMed Central

    Almedom, Ruta B; Liewald, Jana F; Hernando, Guillermina; Schultheis, Christian; Rayes, Diego; Pan, Jie; Schedletzky, Thorsten; Hutter, Harald; Bouzat, Cecilia; Gottschalk, Alexander

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the ‘levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER. PMID:19609303

  1. Structure of the active form of human origin recognition complex and its ATPase motor module

    PubMed Central

    Tocilj, Ante; On, Kin Fan; Yuan, Zuanning; Sun, Jingchuan; Elkayam, Elad; Li, Huilin; Stillman, Bruce; Joshua-Tor, Leemor

    2017-01-01

    Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations. DOI: http://dx.doi.org/10.7554/eLife.20818.001 PMID:28112645

  2. Carbonic anhydrase subunits form a matrix-exposed domain attached to the membrane arm of mitochondrial complex I in plants.

    PubMed

    Sunderhaus, Stephanie; Dudkina, Natalya V; Jänsch, Lothar; Klodmann, Jennifer; Heinemeyer, Jesco; Perales, Mariano; Zabaleta, Eduardo; Boekema, Egbert J; Braun, Hans-Peter

    2006-03-10

    Complex I of Arabidopsis includes five structurally related subunits representing gamma-type carbonic anhydrases termed CA1, CA2, CA3, CAL1, and CAL2. The position of these subunits within complex I was investigated. Direct analysis of isolated subcomplexes of complex I by liquid chromatography linked to tandem mass spectrometry allowed the assignment of the CA subunits to the membrane arm of complex I. Carbonate extraction experiments revealed that CA2 is an integral membrane protein that is protected upon protease treatment of isolated mitoplasts, indicating a location on the matrix-exposed side of the complex. A structural characterization by single particle electron microscopy of complex I from the green alga Polytomella and a previous analysis from Arabidopsis indicate a plant-specific spherical extra-domain of about 60 A in diameter, which is attached to the central part of the membrane arm of complex I on its matrix face. This spherical domain is proposed to contain a heterotrimer of three CA subunits, which are anchored with their C termini to the hydrophobic arm of complex I. Functional implications of the complex I-integrated CA subunits are discussed.

  3. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    PubMed

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion.

  4. Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.

    PubMed

    Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2013-01-01

    Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

  5. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    PubMed Central

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-01-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex. Images PMID:3100577

  6. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    PubMed

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-02-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

  7. RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting

    PubMed Central

    Rajappa-Titu, Lional; Suematsu, Takuma; Munoz-Tello, Paola; Long, Marius; Demir, Özlem; Cheng, Kevin J.; Stagno, Jason R.; Luecke, Hartmut; Amaro, Rommie E.; Aphasizheva, Inna; Aphasizhev, Ruslan; Thore, Stéphane

    2016-01-01

    Terminal uridyltransferases (TUTases) execute 3′ RNA uridylation across protists, fungi, metazoan and plant species. Uridylation plays a particularly prominent role in RNA processing pathways of kinetoplastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei. In mitochondria of this pathogen, most mRNAs are internally modified by U-insertion/deletion editing while guide RNAs and rRNAs are U-tailed. The founding member of TUTase family, RNA editing TUTase 1 (RET1), functions as a subunit of the 3′ processome in uridylation of gRNA precursors and mature guide RNAs. Along with KPAP1 poly(A) polymerase, RET1 also participates in mRNA translational activation. RET1 is divergent from human TUTases and is essential for parasite viability in the mammalian host and the insect vector. Given its robust in vitro activity, RET1 represents an attractive target for trypanocide development. Here, we report high-resolution crystal structures of the RET1 catalytic core alone and in complex with UTP analogs. These structures reveal a tight docking of the conserved nucleotidyl transferase bi-domain module with a RET1-specific C2H2 zinc finger and RNA recognition (RRM) domains. Furthermore, we define RET1 region required for incorporation into the 3′ processome, determinants for RNA binding, subunit oligomerization and processive UTP incorporation, and predict druggable pockets. PMID:27744351

  8. Structural Changes Enable Start Codon Recognition by the Eukaryotic Translation Initiation Complex

    PubMed Central

    Hussain, Tanweer; Llácer, Jose L.; Fernández, Israel S.; Munoz, Antonio; Martin-Marcos, Pilar; Savva, Christos G.; Lorsch, Jon R.; Hinnebusch, Alan G.; Ramakrishnan, V.

    2014-01-01

    Summary During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2α, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon. PMID:25417110

  9. [Generation of the differences of the electric potentials by Rhodospirillum rubrum reaction center complexes devoid of the heavy subunit].

    PubMed

    Barskiĭ, E L; Kondrashin, A A; Samuilov, V D

    1982-10-01

    The electrogenic activity of Rhodospirillum rubrum P870 reaction center complexes devoid of the heavy (H) subunit and retaining the light (L) and medium (M) subunits, was studied. The proteoliposomes containing such reaction center complexes were formed by a self-assembly procedure, using soya bean phospholipids. In the presence of Ca2+ the reaction center proteoliposomes were incorporated into a phospholipid-impregnated Teflon filter separating two solutions of identical composition. After addition of N,N,N',N'-tetra-methyl-p-phenylenediamine (or cytochrome c) and qinone (menadione), illumination caused generation of an electric potential difference between the two filter-separated compartments, the proteoliposome-free compartment being negatively charged. The illuminated proteoliposomes took up penetrating tetraphenylphosphonium cations and, in a lesser degree, tetraphenylborate anions. The data obtained suggest that the reaction center complexes containing only L- and M-subunits possess the electrogenic activity. The H-subunit is not directly involved in membrane potential generation.

  10. Membrane attack complex of complement: distribution of subunits between the hydrocarbon phase of target membranes and water.

    PubMed Central

    Podack, E R; Stoffel, W; Esser, A F; Müller-Eberhard, H J

    1981-01-01

    Membrane destruction by complement is effected by the membrane attack complex (MAC) which is the dimer of a fusion product of the complement proteins C5b, C6, C7, C8, and C9. Phospholipid bilayer vesicles were used as target membranes for the MAC and its intermediate complexes. The subunits of these membrane-bound complexes were explored as to their relative exposure to the hydrocarbon phase of the lipid bilayer and to water surrounding the lipid vesicles. Protein exposed to the aqueous phase was labeled with 125I; protein exposed to the hydrocarbon phase was labeled by using tritiated azido phospholipids and irradiation. Analysis of the membrane-bound MAC showed that subunits C5b, C8 beta, and C9 were exposed to the aqueous phase. The subunits C8 alpha-gamma and C9 were primarily in contact with the hydrocarbon phase. C6 and C7 were little exposed to either phase, suggesting that these proteins are inaccessible within the MAC. Analysis of the intermediate complexes showed that C5b was the subunit most exposed to water in membrane-bound C5b-7, and C5b and C8 beta were the water-exposed subunits in C5b-8. Subunit exposure to the hydrocarbon phase of the lipid bilayer changed during MAC assembly. Whereas all three subunits of C5b-7 carried the phospholipid photolabel; most of the label was bound to the C8 subunit in C5b-8 and to C9 in the MAC. It is proposed that contact with the hydrocarbon core of membranes is established by C5b-7 through each of its subunits, by C5b-8 through C8, and by the MAC through C8 and, particularly, C9. PMID:6270682

  11. Automated recognition and post-coordination of complex clinical terms.

    PubMed

    Gooch, Philip; Roudsari, Abdul

    2011-01-01

    One of the key tasks in integrating guideline-based decision support systems with the electronic patient record is the mapping of clinical terms contained in both guidelines and patient notes to a common, controlled terminology. However, a vocabulary of pre-coordinated terms cannot cover every possible variation - clinical terms are often highly compositional and complex. We present a rule-based approach for automated recognition and post-coordination of clinical terms using minimal, morpheme-based thesauri, neoclassical combining forms and part-of-speech analysis. The process integrates MetaMap with the open-source GATE framework.

  12. Human origin recognition complex is essential for HP1 binding to chromatin and heterochromatin organization

    PubMed Central

    Prasanth, Supriya G.; Shen, Zhen; Prasanth, Kannanganattu V.; Stillman, Bruce

    2010-01-01

    The origin recognition complex (ORC) is a DNA replication initiator protein also known to be involved in diverse cellular functions including gene silencing, sister chromatid cohesion, telomere biology, heterochromatin localization, centromere and centrosome activity, and cytokinesis. We show that, in human cells, multiple ORC subunits associate with hetereochromatin protein 1 (HP1) α- and HP1β-containing heterochromatic foci. Fluorescent bleaching studies indicate that multiple subcomplexes of ORC exist at heterochromatin, with Orc1 stably associating with heterochromatin in G1 phase, whereas other ORC subunits have transient interactions throughout the cell-division cycle. Both Orc1 and Orc3 directly bind to HP1α, and two domains of Orc3, a coiled-coil domain and a mod-interacting region domain, can independently bind to HP1α; however, both are essential for in vivo localization of Orc3 to heterochromatic foci. Direct binding of both Orc1 and Orc3 to HP1 suggests that, after the degradation of Orc1 at the G1/S boundary, Orc3 facilitates assembly of ORC/HP1 proteins to chromatin. Although depletion of Orc2 and Orc3 subunits by siRNA caused loss of HP1α association to heterochromatin, loss of Orc1 and Orc5 caused aberrant HP1α distribution only to pericentric heterochromatin-surrounding nucleoli. Depletion of HP1α from human cells also shows loss of Orc2 binding to heterochromatin, suggesting that ORC and HP1 proteins are mutually required for each other to bind to heterochromatin. Similar to HP1α-depleted cells, Orc2 and Orc3 siRNA-treated cells also show loss of compaction at satellite repeats, suggesting that ORC together with HP1 proteins may be involved in organizing higher-order chromatin structure and centromere function. PMID:20689044

  13. Signal recognition particle receptor is a complex that contains two distinct polypeptide chains

    SciTech Connect

    Tajima, S.; Lauffer, L.; Rath, V.L.; Walter, P.

    1986-10-01

    Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here the authors report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (..cap alpha..-subunit, SR..cap alpha..), a 30-kD ..beta..-subunit SR..beta..). When SRP receptor was purified by SRP-Sepharose affinity chromatography, they observed the co-purification of two other ER membrane proteins. Both proteins are approx.30 kD in size and are immunologically distinct from each other, as well as from SR..cap alpha.. and SRP proteins. One of the 30-kD proteins (SR..beta..) forms a tight complex with SR..cap alpha.. in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR..cap alpha.. or SR..beta... Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. They therefore conclude that SR..beta.. is an integral component of SRP receptor. The presence of SR..beta.. was previously masked by proteolytic breakdown products of SR..cap alpha.. observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR..cap alpha... Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR..cap alpha.. and SR..beta... The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.

  14. Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae

    PubMed Central

    Kawakami, Hironori; Ohashi, Eiji; Tsurimoto, Toshiki; Katayama, Tsutomu

    2016-01-01

    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC. PMID:27148210

  15. Insights into cyclin groove recognition: complex crystal structures and inhibitor design through ligand exchange.

    PubMed

    Kontopidis, George; Andrews, Martin J I; McInnes, Campbell; Cowan, Angela; Powers, Helen; Innes, Lorraine; Plater, Andy; Griffiths, Gary; Paterson, Dougie; Zheleva, Daniella I; Lane, David P; Green, Stephen; Walkinshaw, Malcolm D; Fischer, Peter M

    2003-12-01

    Inhibition of CDK2/CA (cyclin-dependent kinase 2/cyclin A complex) activity through blocking of the substrate recognition site in the cyclin A subunit has been demonstrated to be an effective method for inducing apoptosis in tumor cells. We have used the cyclin binding motif (CBM) present in the tumor suppressor proteins p21(WAF1) and p27(KIP1) as a template to optimize the minimal sequence necessary for CDK2/CA inhibition. A series of peptides were prepared, containing nonnatural amino acids, which possess nano- to micromolar CDK2-inhibitory activity. Here we present X-ray structures of the protein complex CDK2/CA, together with the cyclin groove-bound peptides H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH(2) (peptide 1), H-Arg-Arg-Leu-Ile-Phe-NH(2) (peptide 2), Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH(2) (peptide 3), H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH(2) (peptide 4), and H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH(2) (peptide 5). Some of the peptide complexes presented here were obtained through the novel technique of ligand exchange within protein crystals. This method may find general application for obtaining complex structures of proteins with surface-bound ligands.

  16. {alpha}' Subunit of soybean {beta}-conglycinin forms complex with rice glutelin via a disulphide bond in transgenic rice seeds.

    PubMed

    Motoyama, Takayasu; Maruyama, Nobuyuki; Amari, Yoshiki; Kobayashi, Kanna; Washida, Haruhiko; Higasa, Takahiko; Takaiwa, Fumio; Utsumi, Shigeru

    2009-01-01

    The alpha' and beta subunits of soybean beta-conglycinin were expressed in rice seeds in order to improve the nutritional and physiological properties of rice as a food. The alpha' subunit accumulated in rice seeds at a higher level than the beta subunit, but no detectable difference in mRNA transcription level between subunits was observed. Sequential extraction results indicate that the alpha' subunit formed one or more disulphide bonds with glutelin. Electron microscopic analysis showed that the alpha' subunit and the beta subunit were transported to PB-II together with glutelin. In mature transgenic seeds, the beta subunit accumulated in low electron density regions in the periphery of PB-II, whereas the alpha' subunit accumulated together with glutelin in high-density regions of the periphery. The subcellular localization of mutated alpha' subunits lacking one cysteine residue in the N-terminal mature region (alpha'DeltaCys1) or five cysteine residues in the pro and N-terminal mature regions (alpha'DeltaCys5) were also examined. Low-density regions were formed in PB-II in mature seeds of transgenic rice expressing alpha'DeltaCys 5 and alpha'DeltaCys1. alpha'DeltaCys5 was localized only in the low-density regions, whereas alpha'DeltaCys1 was found in both low- and high-density regions. These results suggest that the alpha' subunit could make a complex via one or more disulphide bonds with glutelin and accumulate together in PB-II of transgenic rice seeds.

  17. Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits

    PubMed Central

    Zhang, Cheng; Sun, Qi; Chen, Rongchang; Chen, Xining; Lin, Jinzhong; Ye, Keqiong

    2016-01-01

    Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1–Utp21, Utp12–Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data. PMID:27330138

  18. Disruptive mitochondrial DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors.

    PubMed

    Gasparre, Giuseppe; Porcelli, Anna Maria; Bonora, Elena; Pennisi, Lucia Fiammetta; Toller, Matteo; Iommarini, Luisa; Ghelli, Anna; Moretti, Massimo; Betts, Christine M; Martinelli, Giuseppe Nicola; Ceroni, Alberto Rinaldi; Curcio, Francesco; Carelli, Valerio; Rugolo, Michela; Tallini, Giovanni; Romeo, Giovanni

    2007-05-22

    Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P=0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.

  19. Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits.

    PubMed

    Zhang, Cheng; Sun, Qi; Chen, Rongchang; Chen, Xining; Lin, Jinzhong; Ye, Keqiong

    2016-09-06

    Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1-Utp21, Utp12-Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data.

  20. Shared Subunits of Tetrahymena Telomerase Holoenzyme and Replication Protein A Have Different Functions in Different Cellular Complexes.

    PubMed

    Upton, Heather E; Chan, Henry; Feigon, Juli; Collins, Kathleen

    2017-01-06

    In most eukaryotes, telomere maintenance relies on telomeric repeat synthesis by a reverse transcriptase named telomerase. To synthesize telomeric repeats, the catalytic subunit telomerase reverse transcriptase (TERT) uses the RNA subunit (TER) as a template. In the ciliate Tetrahymena thermophila, the telomerase holoenzyme consists of TER, TERT, and eight additional proteins, including the telomeric repeat single-stranded DNA-binding protein Teb1 and its heterotrimer partners Teb2 and Teb3. Teb1 is paralogous to the large subunit of the general single-stranded DNA binding heterotrimer replication protein A (RPA). Little is known about the function of Teb2 and Teb3, which are structurally homologous to the RPA middle and small subunits, respectively. Here, epitope-tagging Teb2 and Teb3 expressed at their endogenous gene loci enabled affinity purifications that revealed that, unlike other Tetrahymena telomerase holoenzyme subunits, Teb2 and Teb3 are not telomerase-specific. Teb2 and Teb3 assembled into other heterotrimer complexes, which when recombinantly expressed had the general single-stranded DNA binding activity of RPA complexes, unlike the telomere-specific DNA binding of Teb1 or the TEB heterotrimer of Teb1, Teb2, and Teb3. TEB had no more DNA binding affinity than Teb1 alone. In contrast, heterotrimers reconstituted with Teb2 and Teb3 and two other Tetrahymena RPA large subunit paralogs had higher DNA binding affinity than their large subunit alone. Teb1 and TEB, but not RPA, increased telomerase processivity. We conclude that in the telomerase holoenzyme, instead of binding DNA, Teb2 and Teb3 are Teb1 assembly factors. These findings demonstrate that Tetrahymena telomerase holoenzyme and RPA complexes share subunits and that RPA subunits have distinct functions in different heterotrimer assemblies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Analysis of the mitochondrial encoded subunits of complex I in 20 patients with a complex I deficiency.

    PubMed

    Meulemans, Ann; Lissens, Willy; Van Coster, Rudy; De Meirleir, Linda; Smet, Joél; Nassogne, Marie-Cécile; Liebaers, Inge; Seneca, Sara

    2004-01-01

    NADH-ubiquinone oxidoreductase or complex I deficiency is a frequently diagnosed enzyme defect of the oxidative phosphorylation (OXPHOS) system in humans. However, in many patients, with complex I deficiency and clinical symptoms suggestive of mitochondrial disease, often no genetic defect can be found after investigation of the most common mitochondrial DNA (mtDNA) mutations. In this study, 20 patients were selected with a biochemically documented complex I defect and no common mtDNA mutation. We used the Denaturing Gradient Gel Electrophoresis (DGGE) method with primers encompassing all mitochondrial encoded fragments, to search in a systematic manner for mutations in the mitochondrial genome of complex I. In our group of patients, we were able to detect a total of 96 nucleotide changes. We were not able to find any disease causing mutation in the mitochondrial encoded subunits of complex I. These results suggested that the complex I deficiency in this group of patients is most probably caused by a defect in one of the nuclear encoded structural genes of complex I, or in one of the genes involved in proper assembly of the enzyme.

  2. A formalism for scattering of complex composite structures. I. Applications to branched structures of asymmetric sub-units.

    PubMed

    Svaneborg, Carsten; Pedersen, Jan Skov

    2012-03-14

    We present a formalism for the scattering of an arbitrary linear or acyclic branched structure build by joining mutually non-interacting arbitrary functional sub-units. The formalism consists of three equations expressing the structural scattering in terms of three equations expressing the sub-unit scattering. The structural scattering expressions allow composite structures to be used as sub-units within the formalism itself. This allows the scattering expressions for complex hierarchical structures to be derived with great ease. The formalism is generic in the sense that the scattering due to structural connectivity is completely decoupled from internal structure of the sub-units. This allows sub-units to be replaced by more complex structures. We illustrate the physical interpretation of the formalism diagrammatically. By applying a self-consistency requirement, we derive the pair distributions of an ideal flexible polymer sub-unit. We illustrate the formalism by deriving generic scattering expressions for branched structures such as stars, pom-poms, bottle-brushes, and dendrimers build out of asymmetric two-functional sub-units.

  3. Role of individual subunits of the Neurospora crassa CSN complex in regulation of deneddylation and stability of cullin proteins.

    PubMed

    Wang, Jiyong; Hu, Qiwen; Chen, Huijie; Zhou, Zhipeng; Li, Weihua; Wang, Ying; Li, Shaojie; He, Qun

    2010-12-02

    The Cop9 signalosome (CSN) is an evolutionarily conserved multifunctional complex that controls ubiquitin-dependent protein degradation in eukaryotes. We found seven CSN subunits in Neurospora crassa in a previous study, but only one subunit, CSN-2, was functionally characterized. In this study, we created knockout mutants for the remaining individual CSN subunits in N. crassa. By phenotypic observation, we found that loss of CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, or CSN-7 resulted in severe defects in growth, conidiation, and circadian rhythm; the defect severity was gene-dependent. Unexpectedly, CSN-3 knockout mutants displayed the same phenotype as wild-type N. crassa. Consistent with these phenotypic observations, deneddylation of cullin proteins in csn-1, csn-2, csn-4, csn-5, csn-6, or csn-7 mutants was dramatically impaired, while deletion of csn-3 did not cause any alteration in the neddylation/deneddylation state of cullins. We further demonstrated that CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, and CSN-7, but not CSN-3, were essential for maintaining the stability of Cul1 in SCF complexes and Cul3 and BTB proteins in Cul3-BTB E3s, while five of the CSN subunits, but not CSN-3 and CSN-5, were also required for maintaining the stability of SKP-1 in SCF complexes. All seven CSN subunits were necessary for maintaining the stability of Cul4-DDB1 complexes. In addition, CSN-3 was also required for maintaining the stability of the CSN-2 subunit and FWD-1 in the SCF(FWD-1) complex. Together, these results not only provide functional insights into the different roles of individual subunits in the CSN complex, but also establish a functional framework for understanding the multiple functions of the CSN complex in biological processes.

  4. The structure of FANCL, the catalytic subunit of the Fanconi Anemia core complex

    PubMed Central

    Cole, Ambrose R.; Lewis, Laurence P.C.; Walden, Helen

    2010-01-01

    The Fanconi Anemia pathway is activated in response to DNA damage, leading to monoubiquitination of the substrates FANCI and FANCD2 by the Fanconi Anemia core complex. Here we report the crystal structure of FANCL, the catalytic subunit of the Fanconi Anemia core complex at 3.2 Å. The structure reveals an architecture that is fundamentally different from previous sequence-based predictions. The molecule is composed of an N-terminal E2-like fold, which we term the ELF domain, a novel double-RWD (DRWD) domain, and a C-terminal RING domain predicted to facilitate E2 binding. Binding assays demonstrate that the DRWD domain, but not the ELF domain, is responsible for substrate binding. PMID:20154706

  5. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly

    PubMed Central

    Mueller, Susanne; Wahlander, Asa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy Mankah; Poljak, Kristina; Frey, Alexander D.; Aebi, Markus; Gauss, Robert

    2015-01-01

    Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation. PMID:25995378

  6. A Structure-Based Mechanism for Vesicle Capture by a Multi-Subunit Tethering Complex

    PubMed Central

    Ren, Yi; Yip, Calvin K.; Tripathi, Arati; Huie, David; Jeffrey, Philip D.; Walz, Thomas; Hughson, Frederick M.

    2009-01-01

    Vesicle trafficking requires membrane fusion, mediated by SNARE proteins, and upstream events that probably include “tethering”, an initial long-range attachment between a vesicle and its target organelle. Among the factors proposed to mediate tethering are a set of multisubunit tethering complexes (MTCs). The Dsl1 complex, with only three subunits, is the simplest known MTC, and is essential for the retrograde traffic of COPI-coated vesicles from the Golgi to the ER. To elucidate structural principles underlying MTC function, we have determined the structure of the Dsl1 complex, revealing a tower containing at its base the binding sites for two ER SNAREs and at its tip a flexible lasso for capturing vesicles. The Dsl1 complex binds to individual SNAREs via their N-terminal regulatory domains and also to assembled SNARE complexes; moreover, it is capable of accelerating SNARE complex assembly. Our results suggest that even the simplest MTC may be capable of orchestrating vesicle capture, uncoating, and fusion. PMID:20005805

  7. The origin recognition complex: a biochemical and structural view

    PubMed Central

    Li, Huilin; Stillman, Bruce

    2013-01-01

    The origin recognition complex (ORC) was first discovered in the baker’s yeast in 1992. Identification of ORC opened up a path for subsequent molecular level investigations on how eukaryotic cells initiate and control genome duplication each cell cycle. Twenty years after the first biochemical isolation, ORC is now taking on a three-dimensional shape, although a very blurry shape at the moment, thanks to the recent electron microscopy and image reconstruction efforts. In this chapter, we outline the current biochemical knowledge about ORC from several eukaryotic systems, with emphasis on the most recent structural and biochemical studies. Despite many species-specific properties, an emerging consensus is that ORC is a ATP-dependent machine that recruits other key proteins to form pre-Replicative Complexes (pre-RCs) at many origins of DNA replication, enabling the subsequent initiation of DNA replication in S phase. PMID:22918579

  8. Diversity and Complexity in DNA Recognition by Transcription Factors**

    PubMed Central

    Badis, Gwenael; Berger, Michael F.; Philippakis, Anthony A.; Talukder, Shaheynoor; Gehrke, Andrew R.; Jaeger, Savina A.; Chan, Esther T.; Metzler, Genita; Vedenko, Anastasia; Chen, Xiaoyu; Kuznetsov, Hanna; Wang, Chi-Fong; Coburn, David; Newburger, Daniel E.; Morris, Quaid; Hughes, Timothy R.; Bulyk, Martha L.

    2010-01-01

    Sequence preferences of DNA-binding proteins are a primary mechanism by which cells interpret the genome. Despite these proteins’ central importance in physiology, development, and evolution, comprehensive DNA-binding specificities have been determined experimentally for few proteins. Here, we used microarrays containing all 10-base-pair sequences to examine the binding specificities of 104 distinct mouse DNA-binding proteins representing 22 structural classes. Our results reveal a complex landscape of binding, with virtually every protein analyzed possessing unique preferences. Roughly half of the proteins each recognized multiple distinctly different sequence motifs, challenging our molecular understanding of how proteins interact with their DNA binding sites. This complexity in DNA recognition may be important in gene regulation and in evolution of transcriptional regulatory networks. PMID:19443739

  9. Variations in recollection: The effects of complexity on source recognition

    PubMed Central

    Parks, Colleen M.; Murray, Linda J.; Elfman, Kane; Yonelinas, Andrew P.

    2011-01-01

    Whether recollection is a threshold or signal detection process is highly controversial. U-shaped zROCs observed in tests thought to rely heavily on recollection, such as source memory tests, have provided evidence in favor of the threshold assumption, but zROCs are not always as U-shaped as threshold theory predicts. Source zROCs have been shown to become more linear when the contribution of familiarity to source discriminations is increased, and this may account for the existing results. However, another way in which source zROCs may become more linear is if recollection can become more graded under certain conditions. We tested the ‘graded recollection’ account in the current study. We found that increasing stimulus complexity (i.e., changing from single words to sentences), or increasing source complexity (i.e., changing the sources from audio to videos of speakers), resulted in flatter source zROCs. In addition, conditions expected to reduce recollection (i.e., divided attention and amnesia) had comparable effects on source memory in simple and complex conditions, suggesting that differences between simple and complex conditions were due to differences in the nature of recollection, rather than differences in the utility of familiarity. The results suggest that under conditions of high complexity recollection can appear more graded and it can produce curved ROCs. The results have implications for measurement models and for current theories of recognition memory. PMID:21417513

  10. Chirality Recognition in Camphor - 1,2-PROPANEDIOL Complexes

    NASA Astrophysics Data System (ADS)

    Perez, Cristobal; Fatima, Mariyam; Krin, Anna; Schnell, Melanie

    2017-06-01

    The molecular interactions in complexes involving chiral molecules are of particular interest, because the interactions change in a subtle way upon replacing one of the partners by its mirror image. This is based on the fact that chiral molecules are sensitive probes for other chiral objects and chiral interactions. In this particular case, we will concentrate on molecule-molecule interactions and investigate them with broadband rotational spectroscopy. When two chiral molecules form complexes, the homochiral and heterochiral forms have different structures (and thus rotational constants and spectra) and different energies. They are diastereomers, which can easily be differentiated, for example via molecular spectroscopy. This is often exploited in chemical synthesis for identifying and separating enantiomers. The phenomena involving chirality recognition are relevant in the biosphere, in organic synthesis and in polymer design. We use chirped-pulse Fourier transform microwave (CP-FTMW) spectroscopy to study the structures and the underlying interactions of camphor-1,2-propanediol complexes. This system is also interesting because the complex formation can be expected to be ruled by an interplay between hydrogen bonding to the polar carbonyl group in camphor and dispersion interactions. The spectra are extremely rich because of the high number of conformers for 1,2-propanediol. We started out with racemic mixtures of both camphor and 1,2-propanediol. Using enantiopure samples of different handedness of the two partners nicely simplifies the spectra and guides the assignment. In the talk, we will report on the latest results for this chiral complex.

  11. Structure and Biochemical Properties of Fission Yeast Arp2/3 Complex Lacking the Arp2 Subunit

    SciTech Connect

    Nolen, B.; Pollard, T

    2008-01-01

    Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2? Arp2/3 complex) from fission yeast. An x-ray crystal structure of the ?Arp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2? Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2? Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.

  12. Differential association of protein subunits with the human RNase MRP and RNase P complexes.

    PubMed

    Welting, Tim J M; Kikkert, Bastiaan J; van Venrooij, Walther J; Pruijn, Ger J M

    2006-07-01

    RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60-80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60-80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.

  13. Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement

    SciTech Connect

    Makino, Debora Lika Conti, Elena

    2013-11-01

    The crystallographic steps towards the structure determination of a complete eukaryotic exosome complex bound to RNA are presented. Phasing of this 11-protein subunit complex was carried out via molecular replacement. The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.

  14. Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II*

    PubMed Central

    Maklashina, Elena; Rajagukguk, Sany; Starbird, Chrystal A.; McDonald, W. Hayes; Koganitsky, Anna; Eisenbach, Michael; Iverson, Tina M.; Cecchini, Gary

    2016-01-01

    Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation. PMID:26644464

  15. Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping.

    PubMed

    Clarey, Megan G; Botchan, Michael; Nogales, Eva

    2008-12-01

    Hyperphosphorylation of the Drosophila melanogaster origin recognition complex (DmORC) by cyclin dependent kinases (CDKs) allows nucleotide binding but inhibits the ATPase activity of Orc1, and ablates the ATP-dependent interaction of ORC with DNA. Here we present single particle electron microscopy (EM) studies of ORC bound to nucleotide in both the dephosphorylated and hyper-phosphorylated states. 3D image reconstructions show that nucleotide binding gives rise to an analogous conformation independent of phosphorylation state. At the intermediate resolution achieved in our studies, ATP promotes changes along the toroidal core of the complex with negligible differences contributed by phosphorylation. Thus, hyperphosphorylation of DmORC does not induce meso-scale rearrangement of the ORC structure. To better understand ORC's role in origin remodeling, we performed atomic force microscopy (AFM) studies that show the contour length of a 688bp linear DNA fragment shortens by the equivalent of approximately 130bp upon ORC binding. This data, coupled with previous studies that showed a linking number change in circular DNA upon ORC binding, suggests that ORC may wrap the DNA in a manner akin to DnaA. Based on existing data and our structures, we propose a subunit arrangement for the AAA+ and winged helix domains, and in addition, speculate on a path of the 133bp of DNA around the ORC complex.

  16. The mitochondrial-encoded subunits of respiratory complex I (NADH:ubiquinone oxidoreductase): identifying residues important in mechanism and disease.

    PubMed

    Bridges, Hannah R; Birrell, James A; Hirst, Judy

    2011-06-01

    Complex I (NADH:ubiquinone oxidoreductase) is crucial to respiration in many aerobic organisms. The hydrophilic domain of complex I, containing nine or more redox cofactors, and comprising seven conserved core subunits, protrudes into the mitochondrial matrix or bacterial cytoplasm. The α-helical membrane-bound hydrophobic domain contains a further seven core subunits that are mitochondrial-encoded in eukaryotes and named the ND subunits (ND1-ND6 and ND4L). Complex I couples the oxidation of NADH in the hydrophilic domain to ubiquinone reduction and proton translocation in the hydrophobic domain. Although the mechanisms of NADH oxidation and intramolecular electron transfer are increasingly well understood, the mechanisms of ubiquinone reduction and proton translocation remain only poorly defined. Recently, an α-helical model of the hydrophobic domain of bacterial complex I [Efremov, Baradaran and Sazanov (2010) Nature 465, 441-447] revealed how the 63 transmembrane helices of the seven core subunits are arranged, and thus laid a foundation for the interpretation of functional data and the formulation of mechanistic proposals. In the present paper, we aim to correlate information from sequence analyses, site-directed mutagenesis studies and mutations that have been linked to human diseases, with information from the recent structural model. Thus we aim to identify and discuss residues in the ND subunits of mammalian complex I which are important in catalysis and for maintaining the enzyme's structural and functional integrity.

  17. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

    PubMed Central

    Roignant, Jean-Yves; Treisman, Jessica E.

    2010-01-01

    Summary The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions, and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents Epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII, and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA, but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns. PMID:20946982

  18. Crystal structure of truncated human coatomer protein complex subunit ζ1 (Copζ1).

    PubMed

    Lunev, Sergey; Semmelink, Marije F W; Xian, Jia Ling; Ma, Kai Yu; Leenders, Anna J A; Dömling, Alexander S S; Shtutman, Michael; Groves, Matthew R

    2017-01-01

    The majority of modern anticancer approaches target DNA/protein targets involved in tumour-cell proliferation. Such approaches have a major drawback, as nonproliferating cancer cells remain unaffected and may cause relapse or remission. Human coatomer protein complex I (COPI) subunit ζ (Copζ), a component of the coat protein involved in cell apoptosis and intracellular trafficking, has recently been proposed as a potential anticancer drug target. Previous studies have shown that two different isoforms of the Copζ subunit exist in mammalian cells. While normal cells express both Copζ1 and Copζ2 isoforms, various types of tumour cells display a loss of Copζ2 expression and rely solely on Copζ1 for growth and survival. Subsequent knockdown of Copζ1 results in specific inhibition of both proliferating and dormant tumour-cell populations, with no adverse growth effects on normal cells. Therefore, a Copζ1-targeting therapy was proposed to bypass the problem of dormant cancer cells that are resistant to conventional antiproliferative drugs, which is the major cause of tumour relapse. In order to aid in structure-based inhibitor design, a crystal structure is required. In this article, the recombinant expression, purification, crystallization and crystal structure of Copζ1, as well as the expression and purification of Copζ2, are reported.

  19. Comparative Genomic Analysis of Multi-Subunit Tethering Complexes Demonstrates an Ancient Pan-Eukaryotic Complement and Sculpting in Apicomplexa

    PubMed Central

    Klinger, Christen M.; Klute, Mary J.; Dacks, Joel B.

    2013-01-01

    Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs), factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardiatheta and Bigelowiellanatans. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during, and possibly pre

  20. Comparative genomic analysis of multi-subunit tethering complexes demonstrates an ancient pan-eukaryotic complement and sculpting in Apicomplexa.

    PubMed

    Klinger, Christen M; Klute, Mary J; Dacks, Joel B

    2013-01-01

    Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs), factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardiatheta and Bigelowiellanatans. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during, and possibly pre

  1. Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

    DOE PAGES

    Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; ...

    2014-09-17

    The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells usingmore » variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. In conclusion, these procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.« less

  2. Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

    SciTech Connect

    Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; Hancock, William O.

    2014-09-17

    The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. In conclusion, these procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.

  3. Protein kinase A catalytic subunit primed for action: Time-lapse crystallography of Michaelis complex formation

    DOE PAGES

    Das, Amit; Gerlits, Oksana O.; Parks, Jerry M.; ...

    2015-11-12

    The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg2+ binds first to the M1 site as a complex with ATP and is followed by Mg2+ binding to the M2 site. Furthermore, themore » target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.« less

  4. Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation.

    PubMed

    Das, Amit; Gerlits, Oksana; Parks, Jerry M; Langan, Paul; Kovalevsky, Andrey; Heller, William T

    2015-12-01

    The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg(2+) binds first to the M1 site as a complex with ATP and is followed by Mg(2+) binding to the M2 site. Concurrently, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. Lastly, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.

  5. Shared Protein Complex Subunits Contribute to Explaining Disrupted Co-occurrence

    PubMed Central

    Schneider, Adrian; Seidl, Michael F.; Snel, Berend

    2013-01-01

    The gene composition of present-day genomes has been shaped by a complicated evolutionary history, resulting in diverse distributions of genes across genomes. The pattern of presence and absence of a gene in different genomes is called its phylogenetic profile. It has been shown that proteins whose encoding genes have highly similar profiles tend to be functionally related: As these genes were gained and lost together, their encoded proteins can probably only perform their full function if both are present. However, a large proportion of genes encoding interacting proteins do not have matching profiles. In this study, we analysed one possible reason for this, namely that phylogenetic profiles can be affected by multi-functional proteins such as shared subunits of two or more protein complexes. We found that by considering triplets of proteins, of which one protein is multi-functional, a large fraction of disturbed co-occurrence patterns can be explained. PMID:23874172

  6. Recognition of lipopolysaccharide pattern by TLR4 complexes

    PubMed Central

    Park, Beom Seok; Lee, Jie-Oh

    2013-01-01

    Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. Minute amounts of LPS released from infecting pathogens can initiate potent innate immune responses that prime the immune system against further infection. However, when the LPS response is not properly controlled it can lead to fatal septic shock syndrome. The common structural pattern of LPS in diverse bacterial species is recognized by a cascade of LPS receptors and accessory proteins, LPS binding protein (LBP), CD14 and the Toll-like receptor4 (TLR4)–MD-2 complex. The structures of these proteins account for how our immune system differentiates LPS molecules from structurally similar host molecules. They also provide insights useful for discovery of anti-sepsis drugs. In this review, we summarize these structures and describe the structural basis of LPS recognition by LPS receptors and accessory proteins. PMID:24310172

  7. Structural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin.

    PubMed

    Bourges, Ingrid; Ramus, Claire; Mousson de Camaret, Bénédicte; Beugnot, Réjane; Remacle, Claire; Cardol, Pierre; Hofhaus, Götz; Issartel, Jean-Paul

    2004-11-01

    Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rho degrees cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells, all the seven analysed nuclear-encoded complex I subunits were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I.

  8. Structural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin

    PubMed Central

    2004-01-01

    Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rho° cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells, all the seven analysed nuclear-encoded complex I subunits were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I. PMID:15250827

  9. ALLOSTERY AND SUBSTRATE CHANNELING IN THE TRYPTOPHAN SYNTHASE BIENZYME COMPLEX: EVIDENCE FOR TWO SUBUNIT CONFORMATIONS AND FOUR QUATERNARY STATES

    PubMed Central

    Niks, Dimitri; Hilario, Eduardo; Dierkers, Adam; Ngo, Huu; Borchardt, Dan; Neubauer, Thomas J.; Fan, Li; Mueller, Leonard J.; Dunn, Michael F.

    2014-01-01

    The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. 19F NMR studies of bound α-site substrate analogues, N-(4’-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4’-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of -0.12 ppm. X-ray crystal structures show the F6 and F9 CF3 groups located at the α-β subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in 19F chemical shift results primarily from changes in the α-site ligand conformation. Structures of α-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the α- and β-subunits have closed conformations wherein access of ligands into the α- and β-sites from solution is blocked. Internal and external aldimine structures show the α- and β-subunits with closed and open global conformations, respectively. These results establish that β-subunits exist in two global conformation states, designated open, where the β-sites are freely accessible to substrates, and closed, where the β-site portal into solution is blocked. Switching between these conformations is critically important for the αβ-catalytic cycle. PMID:23952479

  10. Structural insight into the recognition of the H3K4me3 mark by the TFIID subunit TAF3.

    PubMed

    van Ingen, Hugo; van Schaik, Frederik M A; Wienk, Hans; Ballering, Joost; Rehmann, Holger; Dechesne, Annemarie C; Kruijzer, John A W; Liskamp, Rob M J; Timmers, H Th Marc; Boelens, Rolf

    2008-08-06

    Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-pi interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 "methyl-methyl" switch in the histone code dynamically regulates TFIID-promoter association.

  11. Complex I Subunit Gene Therapy With NDUFA6 Ameliorates Neurodegeneration in EAE

    PubMed Central

    Talla, Venu; Koilkonda, Rajeshwari; Porciatti, Vittorio; Chiodo, Vince; Boye, Sanford L.; Hauswirth, William W.; Guy, John

    2015-01-01

    Purpose. To address the permanent disability induced by mitochondrial dysfunction in experimental autoimmune encephalomyelitis (EAE). Methods. Mice sensitized for EAE were rescued by intravitreal injection of adeno-associated viral vector serotype 2 with the complex I subunit gene scAAV-NDUFA6Flag. Controls were injected with a mitochondrially targeted red fluorescent protein (scAAV-COX8-cherry). Another group received scAAV-COX8-cherry, but was not sensitized for EAE. Serial pattern electroretinograms (PERGs) and optical coherent tomography (OCT) evaluated visual function and structure of the retina at 1, 3, and 6 months post injection (MPI). Treated mice were killed 6 MPI for histopathology. Immunodetection of cleaved caspase 3 gauged apoptosis. Complex I activity was assessed spectrophotometrically. Expression of NDUFA6Flag in the retina and optic nerve were evaluated between 1 week to 1 month post injection by RT-PCR, immunofluorescence and immunoblotting. Results. Reverse transcription-PCR and immunoblotting confirmed NDUFA6Flag overexpression with immunoprecipitation and blue native PAGE showing integration into murine complex I. Overexpression of NDUFA6Flag in the visual system of EAE mice rescued retinal complex I activity completely, axonal loss by 73%, and retinal ganglion cell (RGC) loss by 88%, RGC apoptosis by 66%, and restored the 33% loss of complex I activity in EAE to normal levels; thereby, preventing loss of vision indicated by the 43% reduction in the PERG amplitudes of EAE mice. Conclusions. NDUFA6 gene therapy provided long-term suppression of neurodegeneration in the EAE animal model suggesting that it may also ameliorate the mitochondrial dysfunction associated with permanent disability in optic neuritis and MS patients. PMID:25613946

  12. The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity

    PubMed Central

    Angerer, Heike; Radermacher, Michael; Mańkowska, Michalina; Steger, Mirco; Zwicker, Klaus; Heide, Heinrich; Wittig, Ilka; Brandt, Ulrich; Zickermann, Volker

    2014-01-01

    Mitochondrial complex I is the largest and most complicated enzyme of the oxidative phosphorylation system. It comprises a number of so-called accessory subunits of largely unknown structure and function. Here we studied subunit NB4M [NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYRM family of proteins. Chromosomal deletion of the corresponding gene in the yeast Yarrowia lipolytica caused concomitant loss of the mitochondrial acyl carrier protein subunit ACPM1 from the enzyme complex and paralyzed ubiquinone reductase activity. Exchanging the LYR motif and an associated conserved phenylalanine by alanines in subunit NB4M also abolished the activity and binding of subunit ACPM1. We show, by single-particle electron microscopy and structural modeling, that subunits NB4M and ACPM1 form a subdomain that protrudes from the peripheral arm in the vicinity of central subunit domains known to be involved in controlling the catalytic activity of complex I. PMID:24706851

  13. Molecular Recognition in Complexes of TRF Proteins with Telomeric DNA

    PubMed Central

    Wieczór, Miłosz; Tobiszewski, Adrian; Wityk, Paweł; Tomiczek, Bartłomiej; Czub, Jacek

    2014-01-01

    Telomeres are specialized nucleoprotein assemblies that protect the ends of linear chromosomes. In humans and many other species, telomeres consist of tandem TTAGGG repeats bound by a protein complex known as shelterin that remodels telomeric DNA into a protective loop structure and regulates telomere homeostasis. Shelterin recognizes telomeric repeats through its two major components known as Telomere Repeat-Binding Factors, TRF1 and TRF2. These two homologous proteins are therefore essential for the formation and normal function of telomeres. Indeed, TRF1 and TRF2 are implicated in a plethora of different cellular functions and their depletion leads to telomere dysfunction with chromosomal fusions, followed by apoptotic cell death. More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure. Consequently, these proteins are of great interest, not only because of their key role in telomere maintenance and stability, but also as potential drug targets. In the current study, we investigated the molecular basis of telomeric sequence recognition by TRF1 and TRF2 and their DNA binding mechanism. We used molecular dynamics (MD) to calculate the free energy profiles for binding of TRFs to telomeric DNA. We found that the predicted binding free energies were in good agreement with experimental data. Further, different molecular determinants of binding, such as binding enthalpies and entropies, the hydrogen bonding pattern and changes in surface area, were analyzed to decompose and examine the overall binding free energies at the structural level. With this approach, we were able to draw conclusions regarding the consecutive stages of sequence-specific association, and propose a novel aspartate-dependent mechanism of sequence recognition. Finally, our work demonstrates the applicability of computational MD-based methods to studying protein-DNA interactions. PMID:24586793

  14. Subcomplexes of Ancestral Respiratory Complex I Subunits Rapidly Turn Over in Vivo as Productive Assembly Intermediates in Arabidopsis*

    PubMed Central

    Li, Lei; Nelson, Clark J.; Carrie, Chris; Gawryluk, Ryan M. R.; Solheim, Cory; Gray, Michael W.; Whelan, James; Millar, A. Harvey

    2013-01-01

    Subcomplexes of mitochondrial respiratory complex I (CI; EC 1.6.5.3) are shown to turn over in vivo, and we propose a role in an ancestral assembly pathway. By progressively labeling Arabidopsis cell cultures with 15N and isolating mitochondria, we have identified CI subcomplexes through differences in 15N incorporation into their protein subunits. The 200-kDa subcomplex, containing the ancestral γ-carbonic anhydrase (γ-CA), γ-carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CI+CIII2. In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their specific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CI+CIII2, indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a γ-CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occurrence of genes encoding these particular proteins broadly in eukaryotes, except for opisthokonts, provides a framework for the evolutionary conservation of these accessory subunits and evidence of their function in ancestral CI assembly. PMID:23271729

  15. The Not5 subunit of the ccr4-not complex connects transcription and translation.

    PubMed

    Villanyi, Zoltan; Ribaud, Virginie; Kassem, Sari; Panasenko, Olesya O; Pahi, Zoltan; Gupta, Ishaan; Steinmetz, Lars; Boros, Imre; Collart, Martine A

    2014-10-01

    Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

  16. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly.

    PubMed

    Mueller, Susanne; Wahlander, Asa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy Mankah; Poljak, Kristina; Frey, Alexander D; Aebi, Markus; Gauss, Robert

    2015-07-15

    Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation. © 2015 Mueller et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

    DOE PAGES

    Zhang, Chunhua; Brown, Michelle Q.; van de Ven, Wilhelmina; ...

    2015-11-25

    The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells andmore » enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. Ultimately, this study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.« less

  18. Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

    SciTech Connect

    Zhang, Chunhua; Brown, Michelle Q.; van de Ven, Wilhelmina; Zhang, Zhi-Min; Wu, Bin; Young, Michael C.; Synek, Lukáš; Borchardt, Dan; Harrison, Reed; Pan, Songqin; Luo, Nan; Huang, Yu-ming M.; Ghang, Yoo-Jin; Ung, Nolan; Li, Ruixi; Isley, Jonathan; Morikis, Dimitrios; Song, Jikui; Guo, Wei; Hooley, Richard J.; Chang, Chia-en A.; Yang, Zhenbiao; Zarsky, Viktor; Muday, Gloria K.; Hicks, Glenn R.; Raikhel, Natasha V.

    2015-11-25

    The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. Ultimately, this study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

  19. Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

    PubMed Central

    Zhang, Chunhua; Brown, Michelle Q.; van de Ven, Wilhelmina; Zhang, Zhi-Min; Wu, Bin; Young, Michael C.; Synek, Lukáš; Borchardt, Dan; Harrison, Reed; Pan, Songqin; Luo, Nan; Huang, Yu-ming M.; Ghang, Yoo-Jin; Ung, Nolan; Li, Ruixi; Isley, Jonathan; Morikis, Dimitrios; Song, Jikui; Guo, Wei; Hooley, Richard J.; Chang, Chia-en A.; Yang, Zhenbiao; Zarsky, Viktor; Muday, Gloria K.; Hicks, Glenn R.; Raikhel, Natasha V.

    2016-01-01

    The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease. PMID:26607451

  20. Functional Analysis of the Integrator Subunit 12 Identifies a Microdomain That Mediates Activation of the Drosophila Integrator Complex*

    PubMed Central

    Chen, Jiandong; Waltenspiel, Bernhard; Warren, William D.; Wagner, Eric J.

    2013-01-01

    The Drosophila integrator complex consists of 14 subunits that associate with the C terminus of Rpb1 and catalyze the endonucleolytic cleavage of nascent snRNAs near their 3′ ends. Although disruption of almost any integrator subunit causes snRNA misprocessing, very little is known about the role of the individual subunits or the network of structural and functional interactions that exist within the complex. Here we developed an RNAi rescue assay in Drosophila S2 cells to identify functional domains within integrator subunit 12 (IntS12) required for snRNA 3′ end formation. Surprisingly, the defining feature of the Ints12 protein, a highly conserved and centrally located plant homeodomain finger domain, is not required for reporter snRNA 3′ end cleavage. Rather, we find a small, 45-amino acid N-terminal microdomain to be both necessary and nearly sufficient for snRNA biogenesis in cells depleted of endogenous IntS12 protein. This IntS12 microdomain can function autonomously, restoring full integrator processing activity when introduced into a heterologous protein. Moreover, mutations within the microdomain not only disrupt IntS12 function but also abolish binding to other integrator subunits. Finally, the IntS12 microdomain is sufficient to interact and stabilize the putative scaffold integrator subunit, IntS1. Collectively, these results identify an unexpected interaction between the largest and smallest integrator subunits that is essential for the 3′ end formation of Drosophila snRNA. PMID:23288851

  1. Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

    PubMed

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

    2010-01-01

    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

  2. Organelle-specific expression of subunit ND5 of human complex I (NADH dehydrogenase) alters cation homeostasis in Saccharomyces cerevisiae.

    PubMed

    Steffen, Wojtek; Gemperli, Anja C; Cvetesic, Nevena; Steuber, Julia

    2010-09-01

    The ND5 component of the respiratory complex I is a large, hydrophobic subunit encoded by the mitochondrial genome. Its bacterial homologue, the NDH-1 subunit NuoL, acts as a cation transporter in the absence of other NDH-1 subunits. Mutations in human ND5 are frequently observed in neurodegenerative diseases. Wild type and mutant variants of ND5 fused to GFP or a FLAG peptide were targeted to the endoplasmatic reticulum (ER) or the inner mitochondrial membrane of Saccharomyces cerevisiae, which lacks an endogenous complex I. The localization of ND5 fusion proteins was confirmed by microscopic analyses of S. cerevisiae cells, followed by cellular fractionation and immunostaining. The impact of the expression of ND5 fusion proteins on the growth of S. cerevisiae in the presence and absence of added salts was studied. ER-resident ND5 conferred Li(+) sensitivity to S. cerevisiae, which was lost when the E145V variant of ND5 was expressed. All variants of ND5 tested led to increased resistance of S. cerevisiae at high external concentrations of Na(+) or K(+). The data seem to indicate that ND5 influences the salt homeostasis of S. cerevisiae independent of other complex I subunits, and paves the way for functional studies of mutations found in mitochondrially encoded complex I genes.

  3. Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit.

    PubMed

    Suwa, Yoshiaki; Gu, Jianyou; Baranovskiy, Andrey G; Babayeva, Nigar D; Pavlov, Youri I; Tahirov, Tahir H

    2015-06-05

    In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å(2). Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Helicobacter pylori RNA polymerase α-subunit C-terminal domain shows features unique to ɛ-proteobacteria and binds NikR/DNA complexes.

    PubMed

    Borin, Brendan N; Tang, Wei; Krezel, Andrzej M

    2014-04-01

    Bacterial RNA polymerase is a large, multi-subunit enzyme responsible for transcription of genomic information. The C-terminal domain of the α subunit of RNA polymerase (αCTD) functions as a DNA and protein recognition element localizing the polymerase on certain promoter sequences and is essential in all bacteria. Although αCTD is part of RNA polymerase, it is thought to have once been a separate transcription factor, and its primary role is the recruitment of RNA polymerase to various promoters. Despite the conservation of the subunits of RNA polymerase among bacteria, the mechanisms of regulation of transcription vary significantly. We have determined the tertiary structure of Helicobacter pylori αCTD. It is larger than other structurally determined αCTDs due to an extra, highly amphipathic helix near the C-terminal end. Residues within this helix are highly conserved among ɛ-proteobacteria. The surface of the domain that binds A/T rich DNA sequences is conserved and showed binding to DNA similar to αCTDs of other bacteria. Using several NikR dependent promoter sequences, we observed cooperative binding of H. pylori αCTD to NikR:DNA complexes. We also produced αCTD lacking the 19 C-terminal residues, which showed greatly decreased stability, but maintained the core domain structure and binding affinity to NikR:DNA at low temperatures. The modeling of H. pylori αCTD into the context of transcriptional complexes suggests that the additional amphipathic helix mediates interactions with transcriptional regulators.

  5. T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

    PubMed Central

    Manfredi, A A; Protti, M P; Dalton, M W; Howard, J F; Conti-Tronconi, B M

    1993-01-01

    We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected. Images PMID:7688757

  6. Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.

    PubMed

    Worch, Remigiusz; Niedzwiecka, Anna; Stepinski, Janusz; Mazza, Catherine; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Cusack, Stephen; Stolarski, Ryszard

    2005-09-01

    The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.

  7. Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia and failure to thrive.

    PubMed

    Baertling, Fabian; Al-Murshedi, Fathiya; Sánchez-Caballero, Laura; Al-Senaidi, Khalfan; Joshi, Niranjan P; Venselaar, Hanka; van den Brand, Mariël Am; Nijtmans, Leo Gj; Rodenburg, Richard Jt

    2017-03-01

    COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts. This article is protected by copyright. All rights reserved.

  8. PetG and PetN, but not PetL, are essential subunits of the cytochrome b6f complex from Synechocystis PCC 6803.

    PubMed

    Schneider, Dirk; Volkmer, Thomas; Rögner, Matthias

    2007-01-01

    The cytochrome b(6)f complex consists of four large core subunits and an additional four low molecular weight subunits, the function of which is elusive thus far. Here we sought to determine whether small subunits PetG, PetL, and PetN are essential for a cyanobacterial cytochrome b(6)f complex. We found that only PetL is dispensable, whereas PetG and PetN appear to be essential. Possible roles of the small cytochrome b(6)f complex subunits are discussed, and observations from our study are compared with previous findings.

  9. Study of robot landmark recognition with complex background

    NASA Astrophysics Data System (ADS)

    Huang, Yuqing; Yang, Jia

    2007-12-01

    It's of great importance for assisting robot in path planning, position navigating and task performing by perceiving and recognising environment characteristic. To solve the problem of monocular-vision-oriented landmark recognition for mobile intelligent robot marching with complex background, a kind of nested region growing algorithm which fused with transcendental color information and based on current maximum convergence center is proposed, allowing invariance localization to changes in position, scale, rotation, jitters and weather conditions. Firstly, a novel experiment threshold based on RGB vision model is used for the first image segmentation, which allowing some objects and partial scenes with similar color to landmarks also are detected with landmarks together. Secondly, with current maximum convergence center on segmented image as each growing seed point, the above region growing algorithm accordingly starts to establish several Regions of Interest (ROI) orderly. According to shape characteristics, a quick and effectual contour analysis based on primitive element is applied in deciding whether current ROI could be reserved or deleted after each region growing, then each ROI is judged initially and positioned. When the position information as feedback is conveyed to the gray image, the whole landmarks are extracted accurately with the second segmentation on the local image that exclusive to landmark area. Finally, landmarks are recognised by Hopfield neural network. Results issued from experiments on a great number of images with both campus and urban district as background show the effectiveness of the proposed algorithm.

  10. Crystallographic analysis of an RNA polymerase σ-­subunit fragment complexed with −10 promoter element ssDNA: quadruplex formation as a possible tool for engineering crystal contacts in protein–ssDNA complexes

    PubMed Central

    Feklistov, Andrey; Darst, Seth A.

    2013-01-01

    Structural studies of −10 promoter element recognition by domain 2 of the RNA polymerase σ subunit [Feklistov & Darst (2011 ▶), Cell, 147, 1257–1269] reveal an unusual crystal-packing arrangement dominated by G-quartets. The 3′-terminal GGG motif of the oligonucleotide used in crystallization participates in G-quadruplex formation with GGG motifs from symmetry-related complexes. Stacking between neighboring G-quadruplexes results in the formation of pseudo-continuous four-stranded columns running throughout the length of the crystal (G-columns). Here, a new crystal form is presented with a different arrangement of G-columns and it is proposed that the fortuitous finding of G-­quartet packing could be useful in engineering crystal contacts in protein–ssDNA complexes. PMID:23989139

  11. Crystal structure and CRISPR RNA-binding site of the Cmr1 subunit of the Cmr interference complex.

    PubMed

    Sun, Jiali; Jeon, Jae-Hyun; Shin, Minsang; Shin, Ho-Chul; Oh, Byung-Ha; Kim, Jeong-Sun

    2014-02-01

    A multi-subunit ribonucleoprotein complex termed the Cmr RNA-silencing complex recognizes and destroys viral RNA in the CRISPR-mediated immune defence mechanism in many prokaryotes using an as yet unclear mechanism. In Archaeoglobus fulgidus, this complex consists of six subunits, Cmr1-Cmr6. Here, the crystal structure of Cmr1 from A. fulgidus is reported, revealing that the protein is composed of two tightly associated ferredoxin-like domains. The domain located at the N-terminus is structurally most similar to the N-terminal ferredoxin-like domain of the CRISPR RNA-processing enzyme Cas6 from Pyrococcus furiosus. An ensuing mutational analysis identified a highly conserved basic surface patch that binds single-stranded nucleic acids specifically, including the mature CRISPR RNA, but in a sequence-independent manner. In addition, this subunit was found to cleave single-stranded RNA. Together, these studies elucidate the structure and the catalytic activity of the Cmr1 subunit.

  12. The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

    PubMed Central

    Zíková, Alena; Schnaufer, Achim; Dalley, Rachel A.; Panigrahi, Aswini K.; Stuart, Kenneth D.

    2009-01-01

    The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought. PMID:19436713

  13. The Arabidopsis elongator complex subunit2 epigenetically regulates plant immune responses.

    PubMed

    Wang, Yongsheng; An, Chuanfu; Zhang, Xudong; Yao, Jiqiang; Zhang, Yanping; Sun, Yijun; Yu, Fahong; Amador, David Moraga; Mou, Zhonglin

    2013-02-01

    The Arabidopsis thaliana Elongator complex subunit2 (ELP2) genetically interacts with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key transcription coactivator of plant immunity, and regulates the induction kinetics of defense genes. However, the mechanistic relationship between ELP2 and NPR1 and how ELP2 regulates the kinetics of defense gene induction are unclear. Here, we demonstrate that ELP2 is an epigenetic regulator required for pathogen-induced rapid transcriptome reprogramming. We show that ELP2 functions in a transcriptional feed-forward loop regulating both NPR1 and its target genes. An elp2 mutation increases the total methylcytosine number, reduces the average methylation levels of methylcytosines, and alters (increases or decreases) methylation levels of specific methylcytosines. Interestingly, infection of plants with the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000/avrRpt2 induces biphasic changes in DNA methylation levels of NPR1 and PHYTOALEXIN DEFICIENT4 (PAD4), which encodes another key regulator of plant immunity. These dynamic changes are blocked by the elp2 mutation, which is correlated with delayed induction of NPR1 and PAD4. The elp2 mutation also reduces basal histone acetylation levels in the coding regions of several defense genes. Together, our data demonstrate a new role for Elongator in somatic DNA demethylation/methylation and suggest a function for Elongator-mediated chromatin regulation in pathogen-induced transcriptome reprogramming.

  14. Insights into subunit interactions in the Sulfolobus acidocaldarius archaellum cytoplasmic complex.

    PubMed

    Banerjee, Ankan; Neiner, Tomasz; Tripp, Patrick; Albers, Sonja-Verena

    2013-12-01

    Archaella are the archaeal motility structure that is the functional pendant of the bacterial flagellum but is assembled by a mechanism similar to that for type IV pili. Recently, it was shown by Banerjee et al. that FlaX, a crenarchaeal archaellum subunit from Sulfolobus acidocaldarius, forms a ring-like oligomer, and it was proposed that this ring may act as a static platform for torque generation in archaellum rotation [Banerjee A et al. (2012) J Biol Chem 287, 43322-43330]. Moreover, the hexameric crystal structure of FlaI was solved, and its dual function in the assembly and the rotation of the archaellum was demonstrated [Reindl S et al. (2013) Mol Cell 49, 1069-1082]. In this study, we show by biochemical and biophysical techniques that FlaX from S. acidocaldarius acts as a cytoplasmic scaffold in archaellum assembly, as it interacts with FlaI as well as with the recA family protein FlaH, the only cytoplasmic components of the archaellum. Interaction studies using various truncated versions of FlaI demonstrated that its N- and C-termini interact with FlaX. Moreover, using microscale thermophoresis, we show that FlaI, FlaX and FlaH interact with high affinities in the nanomolar range. Therefore, we propose that these three proteins form the cytoplasmic motor complex of the archaellum.

  15. Knockdown of Mediator Complex Subunit 19 Suppresses the Growth and Invasion of Prostate Cancer Cells

    PubMed Central

    Zhao, Hongwei; Lv, Wei; Chen, Jian; Wan, Fengchun; Liu, Dongfu; Gao, Zhenli; Wu, Jitao

    2017-01-01

    Prostate cancer (PCa) is one of the most common cancers in elderly men. Mediator Complex Subunit 19 (Med19) is overexpressed and plays promotional roles in many cancers. However, the roles of Med19 in PCa are still obscure. In this study, by using immunohistochemical staining, we found higher expression level of Med19 in PCa tissues than in adjacent benign prostate tissues. We then knocked down the Med19 expression in PCa cell lines LNCaP and PC3 by using lentivirus siRNA. Cell proliferation, anchor-independent growth, migration, and invasion were suppressed in Med19 knockdown PCa cells. In nude mice xenograft model, we found that Med19 knockdown PCa cells formed smaller tumors with lower proliferation index than did control cells. In the mechanism study, we found that Med19 could regulate genes involved in cell proliferation, cell cycle, and epithelial-mesenchymal transition, including P27, pAKT, pPI3K, IGF1R, E-Cadherin, N-Cadherin, Vimentin, ZEB2, Snail-1 and Snail-2. Targeting Med19 in PCa cells could inhibit the PCa growth and metastasis, and might be a therapeutic option for PCa in the future. PMID:28125713

  16. Purification, preliminary X-ray crystallography and biophysical studies of triose phosphate isomerase-β-globin subunit complex.

    PubMed

    Wahiduzzaman; Dar, Mohammad Aasif; Amir, Mohd; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2017-01-01

    Triose phosphate isomerase (TIM) is a cytoplasmic enzyme of prime importance in the mammalian glycolytic pathway. It has a major role in the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We have successfully purified a stable complex of TIM with β-globin subunit from the sheep kidney using a simple two-step chromatography procedure. It is seen for the first time that TIM is forming a stable complex with β-globin. The purified protein-protein complex was crystallized and preliminary diffraction data were collected at 2.1Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-β-globin complex by monitoring changes in the mean residue ellipticity at 222nm ([θ]222) and difference absorption coefficient at 406nm (Δε406) at pH 7.5 and 25°C. We have observed that GdmCl-induced denaturation is reversible. Coincidence of normalized transition curves of both physical properties ([θ]222 and Δε406) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ]222 and Δε406 were used to estimate the stability parameters of the protein-protein complex. This is the first report on the isolation, purification, crystallization and biophysical characterization of the naturally occurring complex of TIM with the β-globin subunit. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Functional and Topological Analysis of Pen-2, the Fourth Subunit of the γ-Secretase Complex*♦

    PubMed Central

    Bammens, Leen; Chávez-Gutiérrez, Lucía; Tolia, Alexandra; Zwijsen, An; De Strooper, Bart

    2011-01-01

    The γ-secretase complex is a member of the family of intramembrane cleaving proteases, involved in the generation of the Aβ peptides in Alzheimer disease. One of the four subunits of the complex, presenilin, harbors the catalytic site, although the role of the other three subunits is less well understood. Here, we studied the role of the smallest subunit, Pen-2, in vivo and in vitro. We found a profound Notch-deficiency phenotype in Pen-2−/− embryos confirming the essential role of Pen-2 in the γ-secretase complex. We used Pen-2−/− fibroblasts to investigate the structure-function relation of Pen-2 by the scanning cysteine accessibility method. We showed that glycine 22 and proline 27 in hydrophobic domain 1 of Pen-2 are essential for complex formation and stability of γ-secretase. We also demonstrated that hydrophobic domain 1 and the loop domain of Pen-2 are located in a water-containing cavity and are in short proximity to the presenilin C-terminal fragment. We finally demonstrated the essential role of Pen-2 for the proteolytic activity of the complex. Our study supports the hypothesis that Pen-2 is more than a structural component of the γ-secretase complex and may contribute to the catalytic mechanism of the enzyme. PMID:21296884

  18. The origin of the supernumerary subunits and assembly factors of complex I: A treasure trove of pathway evolution.

    PubMed

    Elurbe, Dei M; Huynen, Martijn A

    2016-07-01

    We review and document the evolutionary origin of all complex I assembly factors and nine supernumerary subunits from protein families. Based on experimental data and the conservation of critical residues we identify a spectrum of protein function conservation between the complex I representatives and their non-complex I homologs. This spectrum ranges from proteins that have retained their molecular function but in which the substrate specificity may have changed or have become more specific, like NDUFAF5, to proteins that have lost their original molecular function and critical catalytic residues like NDUFAF6. In between are proteins that have retained their molecular function, which however appears unrelated to complex I, like ACAD9, or proteins in which amino acids of the active site are conserved but for which no enzymatic activity has been reported, like NDUFA10. We interpret complex I evolution against the background of molecular evolution theory. Complex I supernumerary subunits and assembly factors appear to have been recruited from proteins that are mitochondrial and/or that are expressed when complex I is active. Within the evolution of complex I and its assembly there are many cases of neofunctionalization after gene duplication, like ACAD9 and TMEM126B, one case of subfunctionalization: ACPM1 and ACPM2 in Yarrowia lipolytica, and one case in which a complex I protein itself appears to have been the source of a new protein from another complex: NDUFS6 gave rise to cytochrome c oxidase subunit COX4/COX5b. Complex I and its assembly can therewith be regarded as a treasure trove for pathway evolution. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

  19. X-ray structures of the three Lactococcus lactis dihydroxyacetone kinase subunits and of a transient intersubunit complex.

    PubMed

    Zurbriggen, Andreas; Jeckelmann, Jean-Marc; Christen, Sandra; Bieniossek, Christoph; Baumann, Ulrich; Erni, Bernhard

    2008-12-19

    Bacterial dihydroxyacetone (Dha) kinases do not exchange the ADP for ATP but utilize a subunit of the phosphoenolpyruvate carbohydrate phosphotransferase system for in situ rephosphorylation of a permanently bound ADP-cofactor. Here we report the 2.1-angstroms crystal structure of the transient complex between the phosphotransferase subunit DhaM of the phosphotransferase system and the nucleotide binding subunit DhaL of the Dha kinase of Lactococcus lactis, the 1.1-angstroms structure of the free DhaM dimer, and the 2.5-angstroms structure of the Dha-binding DhaK subunit. Conserved salt bridges and an edge-to-plane stacking contact between two tyrosines serve to orient DhaL relative to the DhaM dimer. The distance between the imidazole Nepsilon2 of the DhaM His-10 and the beta-phosphate oxygen of ADP, between which the gamma-phosphate is transferred, is 4.9 angstroms. An invariant arginine, which is essential for activity, is appropriately positioned to stabilize the gamma-phosphate in the transition state. The (betaalpha)4alpha fold of DhaM occurs a second time as a subfold in the DhaK subunit. By docking DhaL-ADP to this subfold, the nucleotide bound to DhaL and the C1-hydroxyl of Dha bound to DhaK are positioned for in-line transfer of phosphate.

  20. Identification of conserved, centrosome-targeting ASH domains in TRAPPII complex subunits and TRAPPC8

    PubMed Central

    2014-01-01

    Background Assembly of primary cilia relies on vesicular trafficking towards the cilium base and intraflagellar transport (IFT) between the base and distal tip of the cilium. Recent studies have identified several key regulators of these processes, including Rab GTPases such as Rab8 and Rab11, the Rab8 guanine nucleotide exchange factor Rabin8, and the transport protein particle (TRAPP) components TRAPPC3, -C9, and -C10, which physically interact with each other and function together with Bardet Biedl syndrome (BBS) proteins in ciliary membrane biogenesis. However, despite recent advances, the exact molecular mechanisms by which these proteins interact and target to the basal body to promote ciliogenesis are not fully understood. Results We surveyed the human proteome for novel ASPM, SPD-2, Hydin (ASH) domain-containing proteins. We identified the TRAPP complex subunits TRAPPC8, -9, -10, -11, and -13 as novel ASH domain-containing proteins. In addition to a C-terminal ASH domain region, we predict that the N-terminus of TRAPPC8, -9, -10, and -11, as well as their yeast counterparts, consists of an α-solenoid bearing stretches of multiple tetratricopeptide (TPR) repeats. Immunofluorescence microscopy analysis of cultured mammalian cells revealed that exogenously expressed ASH domains, as well as endogenous TRAPPC8, localize to the centrosome/basal body. Further, depletion of TRAPPC8 impaired ciliogenesis and GFP-Rabin8 centrosome targeting. Conclusions Our results suggest that ASH domains confer targeting to the centrosome and cilia, and that TRAPPC8 has cilia-related functions. Further, we propose that the yeast TRAPPII complex and its mammalian counterpart are evolutionarily related to the bacterial periplasmic trafficking chaperone PapD of the usher pili assembly machinery. PMID:25018876

  1. Isolation of photosystem II-enriched membranes and the oxygen-evolving complex subunit proteins from higher plants.

    PubMed

    Yamamoto, Yasusi; Leng, Jing; Shen, Jian-Ren

    2011-01-01

    We describe methods to isolate highly active oxygen-evolving photosystem II (PSII) membranes and core complexes from higher plants, and to purify subunits of the oxygen-evolving complex (OEC). The membrane samples used as the material for various in vitro studies of PSII are prepared by solubilizing thylakoid membranes with the nonionic detergent Triton X-100, and the core complexes are prepared by further solubilization of the PSII membranes with n-dodecyl-β-D-maltoside (β-DDM). The OEC subunit proteins are dissociated from the PSII-enriched membranes by alkaline or salt treatment, and are then purified by ion-exchange chromatography using an automated high performance liquid chromatography system.

  2. Do nuclear-encoded core subunits of mitochondrial complex I confer genetic susceptibility to schizophrenia in Han Chinese populations?

    PubMed

    Li, Xiao; Zhang, Wen; Tang, Jinsong; Tan, Liwen; Luo, Xiong-jian; Chen, Xiaogang; Yao, Yong-Gang

    2015-06-08

    Schizophrenia is one of the most prevalent psychiatric disorders with complex genetic etiology. Accumulating evidence suggests that energy metabolism and oxidative stress play important roles in the pathophysiology of schizophrenia. Dysfunction of mitochondrial respiratory chain and altered expression of complex I subunits were frequently reported in schizophrenia. To investigate whether nuclear-encoded core subunit genes of mitochondrial complex I are associated with schizophrenia, we performed a genetic association study in Han Chinese. In total, 46 tag single nucleotide polymorphisms (SNPs) from 7 nuclear-encoded core genes of mitochondrial complex I were genotyped in 918 schizophrenia patients and 1042 healthy controls. We also analyzed these SNPs in a large sample mainly composed of Europeans through using the available GWAS datasets from the Psychiatric Genomics Consortium (PGC). No significant associations were detected between these SNPs and schizophrenia in Han Chinese and the PGC data set. However, we observed nominal significant associations of 2 SNPs in the NDUFS1 gene and 4 SNPs in the NDUFS2 gene with early onset schizophrenia (EOS), but none of these associations survived the Bonferroni correction. Taken together, our results suggested that common SNPs in the nuclear-encoded core subunit genes of mitochondrial complex I may not confer genetic susceptibility to schizophrenia.

  3. Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE).

    PubMed

    Wu, Jinzi; Luo, Xiaoting; Yan, Liang-Jun

    2015-01-01

    The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional non-gradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.

  4. Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits.

    PubMed

    Kubota, Hisako; Sakurai, Isamu; Katayama, Kenta; Mizusawa, Naoki; Ohashi, Shunsuke; Kobayashi, Masami; Zhang, Pengpeng; Aro, Eva-Mari; Wada, Hajime

    2010-01-01

    We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni2+-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b(6)f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.

  5. Novel RNA-binding properties of Pop3p support a role for eukaryotic RNase P protein subunits in substrate recognition.

    PubMed

    Brusca, E M; True, H L; Celander, D W

    2001-11-09

    Ribonuclease P (RNase P) catalyzes the 5'-end maturation of transfer RNA molecules. Recent evidence suggests that the eukaryotic protein subunits may provide substrate-binding functions (True, H. L., and Celander, D. W. (1998) J. Biol. Chem. 273, 7193-7196). We now report that Pop3p, an essential protein subunit of the holoenzyme in Saccharomyces cerevisiae, displays novel RNA-binding properties. A recombinant form of Pop3p (H6Pop3p) displays a 3-fold greater affinity for binding pre-tRNA substrates relative to tRNA products. The recognition sequence for the H6Pop3p-substrate interaction in vitro was mapped to a 39-nucleotide long sequence that extends from position -21 to +18 surrounding the natural processing site in pre-tRNA substrates. H6Pop3p binds a variety of RNA molecules with high affinity (K(d) = 16-25 nm) and displays a preference for single-stranded RNAs. Removal or modification of basic C-terminal residues attenuates the RNA-binding properties displayed by the protein specifically for a pre-tRNA substrate. These studies support the model that eukaryotic RNase P proteins bind simultaneously to the RNA subunit and RNA substrate.

  6. Cdc73 subunit of the Paf1 complex contains a C-terminal Ras-like domain that promotes association of Paf1 complex with chromatin

    SciTech Connect

    Amrich C. G.; Heroux A.; Davis, C. P.; Rogal, W. P.; Shirra, M. K.; Gardner, R. G.; Arndt, K. M.; VanDemark, A. P.

    2012-03-30

    The conserved Paf1 complex localizes to the coding regions of genes and facilitates multiple processes during transcription elongation, including the regulation of histone modifications. However, the mechanisms that govern Paf1 complex recruitment to active genes are undefined. Here we describe a previously unrecognized domain within the Cdc73 subunit of the Paf1 complex, the Cdc73 C-domain, and demonstrate its importance for Paf1 complex occupancy on transcribed chromatin. Deletion of the C-domain causes phenotypes associated with elongation defects without an apparent loss of complex integrity. Simultaneous mutation of the C-domain and another subunit of the Paf1 complex, Rtf1, causes enhanced mutant phenotypes and loss of histone H3 lysine 36 trimethylation. The crystal structure of the C-domain reveals unexpected similarity to the Ras family of small GTPases. Instead of a deep nucleotide-binding pocket, the C-domain contains a large but comparatively flat surface of highly conserved residues, devoid of ligand. Deletion of the C-domain results in reduced chromatin association for multiple Paf1 complex subunits. We conclude that the Cdc73 C-domain probably constitutes a protein interaction surface that functions with Rtf1 in coupling the Paf1 complex to the RNA polymerase II elongation machinery.

  7. Complex regulation of γ-secretase: from obligatory to modulatory subunits

    PubMed Central

    Gertsik, Natalya; Chiu, Danica; Li, Yue-Ming

    2014-01-01

    γ-Secretase is a four subunit, 19-pass transmembrane enzyme that cleaves amyloid precursor protein (APP), catalyzing the formation of amyloid beta (Aβ) peptides that form amyloid plaques, which contribute to Alzheimer’s disease (AD) pathogenesis. γ-Secretase also cleaves Notch, among many other type I transmembrane substrates. Despite its seemingly promiscuous enzymatic capacity, γ-secretase activity is tightly regulated. This regulation is a function of many cellular entities, including but not limited to the essential γ-secretase subunits, nonessential (modulatory) subunits, and γ-secretase substrates. Regulation is also accomplished by an array of cellular events, such as presenilin (active subunit of γ-secretase) endoproteolysis and hypoxia. In this review we discuss how γ-secretase is regulated with the hope that an advanced understanding of these mechanisms will aid in the development of effective therapeutics for γ-secretase-associated diseases like AD and Notch-addicted cancer. PMID:25610395

  8. Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase

    PubMed Central

    Mittag, Tanja; Marsh, Joseph; Grishaev, Alexander; Orlicky, Stephen; Lin, Hong; Sicheri, Frank; Tyers, Mike; Forman-Kay, Julie D.

    2010-01-01

    Summary Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations utilizing experimental NMR and small-angle x-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated. PMID:20399186

  9. Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase.

    PubMed

    Mittag, Tanja; Marsh, Joseph; Grishaev, Alexander; Orlicky, Stephen; Lin, Hong; Sicheri, Frank; Tyers, Mike; Forman-Kay, Julie D

    2010-03-14

    Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. Structure/Function Implications in a Dynamic Complex of the Intrinsically Disordered Sic1 with the Cdc4 Subunit of an SCF Ubiquitin Ligase

    SciTech Connect

    Mittag, Tanja; Marsh, Joseph; Grishaev, Alexander; Orlicky, Stephen; Lin, Hong; Sicheri, Frank; Tyers, Mike; Forman-Kay, Julie D.

    2010-11-22

    Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.

  11. The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

    PubMed Central

    2014-01-01

    Background The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. Findings This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. Conclusions In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle. PMID:25379053

  12. Concerted interaction between origin recognition complex (ORC), nucleosomes and replication origin DNA ensures stable ORC-origin binding.

    PubMed

    Hizume, Kohji; Yagura, Masaru; Araki, Hiroyuki

    2013-09-01

    Chromosomal replication origins, where DNA replication is initiated, are determined in eukaryotic cells by specific binding of a six-subunit origin recognition complex (ORC). Many biochemical analyses have showed the detailed properties of the ORC-DNA interaction. However, because of the lack of in vitro analysis, the molecular architecture of the ORC-chromatin interaction is unclear. Recently, mainly from in vivo analyses, a role of chromatin in the ORC-origin interaction has been reported, including the existence of a specific pattern of nucleosome positioning around origins and of a specific interaction between chromatin-or core histones-and Orc1, a subunit of ORC. Therefore, to understand how ORC establishes its interaction with origin in vivo, it is essential to know the molecular mechanisms of the ORC-chromatin interaction. Here, we show that ORC purified from yeast binds more stably to origin-containing reconstituted chromatin than to naked DNA and forms a nucleosome-free region at origins. Molecular imaging using atomic force microscopy (AFM) shows that ORC associates with the adjacent nucleosomes and forms a larger complex. Moreover, stable binding of ORC to chromatin requires linker DNA. Thus, ORC establishes its interaction with origin by binding to both nucleosome-free origin DNA and neighboring nucleosomes. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  13. Systematic expression analysis of the mitochondrial complex III subunits identifies UQCRC1 as biomarker in clear cell renal cell carcinoma

    PubMed Central

    Ellinger, Jörg; Gromes, Arabella; Poss, Mirjam; Brüggemann, Maria; Schmidt, Doris; Ellinger, Nadja; Tolkach, Yuri; Dietrich, Dimo; Kristiansen, Glen; Müller, Stefan C.

    2016-01-01

    Mitochondrial dysfunction is common in cancer, and the mitochondrial electron transport chain is often affected in carcinogenesis. So far, few is known about the expression of the mitochondrial complex III (ubiquinol-cytochrome c reductase complex) subunits in clear cell renal cell carcinoma (ccRCC). In this study, the NextBio database was used to determine an expression profile of the mitochondrial complex III subunits based on published microarray studies. We observed that five out of 11 subunits of the complex III were downregulated in at least three microarray studies. The decreased mRNA expression level of UQCRFS1 and UQCRC1 in ccRCC was confirmed using PCR. Low mRNA levels UQCRC1 were also correlated with a shorter period of cancer-specific and overall survival. Furthermore, UQCRFS1 and UQCRC1 were also decreased in ccRCC on the protein level as determined using Western blotting and immunohistochemistry. UQCRC1 protein expression was also lower in ccRCC than in papillary and chromophobe subtypes. Analyzing gene expression and DNA methylation in The Cancer Genome Atlas cohort revealed an inverse correlation of gene expression and DNA methylation, suggesting that DNA hypermethylation is regulating the expression of UQCRC1 and UQCRFS1. Taken together, our data implicate that dysregulated UQCRC1 and UQCRFS1 are involved in impaired mitochondrial electron transport chain function. PMID:27845902

  14. Mitochondrial proteome analysis reveals depression of the Ndufs3 subunit and activity of complex I in diabetic rat brain.

    PubMed

    Taurino, Federica; Stanca, Eleonora; Siculella, Luisa; Trentadue, Raffaella; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2012-04-18

    Type-1 diabetes resulting from defective insulin secretion and consequent hyperglycemia, is associated with "diabetic encephalopathy." This is characterized by brain neurophysiological and structural changes resulting in impairment of cognitive function. The present proteomic analysis of brain mitochondrial proteins from streptozotocin-induced type-1 diabetic rats, shows a large decrement of the Ndufs3 protein subunit of complex I, decreased level of the mRNA and impaired catalytic activity of the complex in the diabetic rats as compared to controls. The severe depression of the expression and enzymatic activity of complex I can represent a critical contributing factor to the onset of the diabetic encephalopathy in type-1 diabetes.

  15. Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.

    PubMed

    Cafaro, Valeria; Scognamiglio, Roberta; Viggiani, Ambra; Izzo, Viviana; Passaro, Irene; Notomista, Eugenio; Piaz, Fabrizio Dal; Amoresano, Angela; Casbarra, Annarita; Pucci, Piero; Di Donato, Alberto

    2002-11-01

    This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.

  16. The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.

    PubMed

    Singh, Dhirendra; Sielaff, Hendrik; Sundararaman, Lavanya; Bhushan, Shashi; Grüber, Gerhard

    2016-02-01

    A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s(-1) and 30.4 s(-1), respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis.

  17. Structure of a specific peptide complex of the carboxy-terminal SH2 domain from the p85 alpha subunit of phosphatidylinositol 3-kinase.

    PubMed Central

    Breeze, A L; Kara, B V; Barratt, D G; Anderson, M; Smith, J C; Luke, R W; Best, J R; Cartlidge, S A

    1996-01-01

    We have determined the solution structure of the C-terminal SH2 domain of the p85 alpha subunit of human phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) in complex with a phosphorylated tyrosine pentapeptide sequence from the platelet-derived growth factor receptor using heteronuclear nuclear magnetic resonance spectroscopy. Overall, the structure is similar to other SH2 domain complexes, but displays different detail interactions within the phosphotyrosine binding site and in the recognition site for the +3 methionine residue of the peptide, the side chain of which inserts into a particularly deep and narrow pocket which is displaced relative to that of other SH2 domains. The contacts made within this +3 pocket provide the structural basis for the strong selection for methionine at this position which characterizes the SH2 domains of PI3-kinase. Comparison with spectral and structural features of the uncomplexed domain shows that the long BG loop becomes less mobile in the presence of the bound peptide. In contrast, extreme resonance broadening encountered for most residues in the beta D', beta E and beta F strands and associated connecting loops of the domain in the absence of peptide persists in the complex, implying conformational averaging in this part of the molecule on a microsecond-to-millisecond time scale. Images PMID:8670861

  18. The eIF3 complex of Leishmania-subunit composition and mode of recruitment to different cap-binding complexes.

    PubMed

    Meleppattu, Shimi; Kamus-Elimeleh, Dikla; Zinoviev, Alexandra; Cohen-Mor, Shahar; Orr, Irit; Shapira, Michal

    2015-07-27

    Eukaryotic initiation factor 3 (eIF3) is a multi-protein complex and a key participant in the assembly of the translation initiation machinery. In mammals, eIF3 comprises 13 subunits, most of which are characterized by conserved structural domains. The trypanosomatid eIF3 subunits are poorly conserved. Here, we identify 12 subunits that comprise the Leishmania eIF3 complex (LeishIF3a-l) by combining bioinformatics with affinity purification and mass spectrometry analyses. These results highlight the strong association of LeishIF3 with LeishIF1, LeishIF2 and LeishIF5, suggesting the existence of a multi-factor complex. In trypanosomatids, the translation machinery is tightly regulated in the different life stages of these organisms as part of their adaptation and survival in changing environments. We, therefore, addressed the mechanism by which LeishIF3 is recruited to different mRNA cap-binding complexes. A direct interaction was observed in vitro between the fully assembled LeishIF3 complex and recombinant LeishIF4G3, the canonical scaffolding protein of the cap-binding complex in Leishmania promastigotes. We further highlight a novel interaction between the C-terminus of LeishIF3a and LeishIF4E1, the only cap-binding protein that efficiently binds the cap structure under heat shock conditions, anchoring a complex that is deficient of any MIF4G-based scaffolding subunit. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. The eIF3 complex of Leishmania—subunit composition and mode of recruitment to different cap-binding complexes

    PubMed Central

    Meleppattu, Shimi; Kamus-Elimeleh, Dikla; Zinoviev, Alexandra; Cohen-Mor, Shahar; Orr, Irit; Shapira, Michal

    2015-01-01

    Eukaryotic initiation factor 3 (eIF3) is a multi-protein complex and a key participant in the assembly of the translation initiation machinery. In mammals, eIF3 comprises 13 subunits, most of which are characterized by conserved structural domains. The trypanosomatid eIF3 subunits are poorly conserved. Here, we identify 12 subunits that comprise the Leishmania eIF3 complex (LeishIF3a-l) by combining bioinformatics with affinity purification and mass spectrometry analyses. These results highlight the strong association of LeishIF3 with LeishIF1, LeishIF2 and LeishIF5, suggesting the existence of a multi-factor complex. In trypanosomatids, the translation machinery is tightly regulated in the different life stages of these organisms as part of their adaptation and survival in changing environments. We, therefore, addressed the mechanism by which LeishIF3 is recruited to different mRNA cap-binding complexes. A direct interaction was observed in vitro between the fully assembled LeishIF3 complex and recombinant LeishIF4G3, the canonical scaffolding protein of the cap-binding complex in Leishmania promastigotes. We further highlight a novel interaction between the C-terminus of LeishIF3a and LeishIF4E1, the only cap-binding protein that efficiently binds the cap structure under heat shock conditions, anchoring a complex that is deficient of any MIF4G-based scaffolding subunit. PMID:26092695

  20. Insights into Degron Recognition by APC/C Coactivators from the Structure of an Acm1-Cdh1 Complex

    PubMed Central

    He, Jun; Chao, William C.H.; Zhang, Ziguo; Yang, Jing; Cronin, Nora; Barford, David

    2013-01-01

    Summary The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs—predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor. PMID:23707760

  1. The location of NuoL and NuoM subunits in the membrane domain of the Escherichia coli complex I: implications for the mechanism of proton pumping.

    PubMed

    Holt, Peter J; Morgan, David J; Sazanov, Leonid A

    2003-10-31

    The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains. In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents. Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex. Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex. The presence of N,N-dimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits. Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide. A model of subunit arrangement in bacterial complex I based on these data is proposed. Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm. This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes.

  2. A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation

    PubMed Central

    Bleichert, Franziska; Balasov, Maxim; Chesnokov, Igor; Nogales, Eva; Botchan, Michael R; Berger, James M

    2013-01-01

    In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2–7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI: http://dx.doi.org/10.7554/eLife.00882.001 PMID:24137536

  3. A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation.

    PubMed

    Bleichert, Franziska; Balasov, Maxim; Chesnokov, Igor; Nogales, Eva; Botchan, Michael R; Berger, James M

    2013-10-08

    In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2-7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI:http://dx.doi.org/10.7554/eLife.00882.001.

  4. Cellular senescence regulated by SWI/SNF complex subunits through p53/p21 and p16/pRB pathway.

    PubMed

    He, Ling; Chen, Ying; Feng, Jianguo; Sun, Weichao; Li, Shun; Ou, Mengting; Tang, Liling

    2017-09-01

    SWI/SNF complex is an evolutionarily well-conserved chromatin-remodeling complex, which is implicated in the nucleosomes removing or sliding, impacting on the DNA repair, replication and genes expression regulation. The SWI/SNF complex consists up to 12 protein subunits. The catalytic subunits are BRG1 or BRM, which are exclusive ATPase subunits. BRG1 has been reported to play an important role in cellular senescence. However, The function of non-catalytic subunits involved in cellular senescence is rarely investigated. Therefore, we focused on the senescence regulation roles of SWI/SNF non-catalytic subunits in cellular senescent model induced by H2O2. H2O2 treatment was used to induce cellular senescence models in vitro. Screening the candidate subunits involved in this process by comparing the expression levels of SWI/SNF subunits with/without H2O2 treatment. Over-expression and knockdown the candidate subunits were utilized to investigate the functions and mechanism of the subunits involved in senescence regulation. The expressions of BAF57, BAF60a and SNF5 were changed significantly after H2O2 treatment. Overexpression of the three subunits separately induced cell growth arrest in both HaCaT and GLL19 cells, while knockdown of the subunits separately eased the senescence induced by H2O2 treatment. Results further showed that BAF57, BAF60a and SNF5 regulated cellular senescence via both p53/p21 and p16/pRB pathways, and the three subunits all had a directly interaction with p53. These results indicated that BAF57, BAF60a and SNF5 might act as novel pro-senescence factors in both normal and tumor human skin cells. Therefore, inhibiting expression of the three factors might delay the cellular senescence process. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. A novel multi-view object recognition in complex background

    NASA Astrophysics Data System (ADS)

    Chang, Yongxin; Yu, Huapeng; Xu, Zhiyong; Fu, Chengyu; Gao, Chunming

    2015-02-01

    Recognizing objects from arbitrary aspects is always a highly challenging problem in computer vision, and most existing algorithms mainly focus on a specific viewpoint research. Hence, in this paper we present a novel recognizing framework based on hierarchical representation, part-based method and learning in order to recognize objects from different viewpoints. The learning evaluates the model's mistakes and feeds it back the detector to avid the same mistakes in the future. The principal idea is to extract intrinsic viewpoint invariant features from the unseen poses of object, and then to take advantage of these shared appearance features to support recognition combining with the improved multiple view model. Compared with other recognition models, the proposed approach can efficiently tackle multi-view problem and promote the recognition versatility of our system. For an quantitative valuation The novel algorithm has been tested on several benchmark datasets such as Caltech 101 and PASCAL VOC 2010. The experimental results validate that our approach can recognize objects more precisely and the performance outperforms others single view recognition methods.

  6. Shape Recognition Of Complex Objects By Syntactical Primitives

    NASA Astrophysics Data System (ADS)

    Lenger, D.; Cipovic, H.

    1985-04-01

    The paper describes a pattern recognition method based on syntactic image analysis applicable in autonomous systems of robot vision for the purpose of pattern detection or classification. The discrimination of syntactic elements is realized by polygonal approximation of contours employing a very fast algorithm based upon coding, local pixel logic and methods of choice instead of numerical methods. Semantic information is derived from attributes calculated from the filtered shape vector. No a priori information on image objects is required, and the choice of starting point is determined by finding the significant directions on the shape vector. The radius of recognition sphere is minimum Euclidian distance, i.e. maximum similarity between the unknown model and each individual grammar created in the learning phase. By keeping information on derivations of individual syntactic elements, an alternative of parsing recognition is left. The analysis is very flexible, and permits the recognition of highly distorted or even partially visible objects. The output from syntactic analyzer is the measure of irregularity, and the method is thus applicable in any application where sample deformation is being examined.

  7. Spontaneous Object Recognition Memory in Aged Rats: Complexity versus Similarity

    ERIC Educational Resources Information Center

    Gamiz, Fernando; Gallo, Milagros

    2012-01-01

    Previous work on the effect of aging on spontaneous object recognition (SOR) memory tasks in rats has yielded controversial results. Although the results at long-retention intervals are consistent, conflicting results have been reported at shorter delays. We have assessed the potential relevance of the type of object used in the performance of…

  8. Spontaneous Object Recognition Memory in Aged Rats: Complexity versus Similarity

    ERIC Educational Resources Information Center

    Gamiz, Fernando; Gallo, Milagros

    2012-01-01

    Previous work on the effect of aging on spontaneous object recognition (SOR) memory tasks in rats has yielded controversial results. Although the results at long-retention intervals are consistent, conflicting results have been reported at shorter delays. We have assessed the potential relevance of the type of object used in the performance of…

  9. Characterization of the interaction between subunits of the botulinum toxin complex produced by serotype D through tryptic susceptibility of the isolated components and complex forms.

    PubMed

    Suzuki, Tomonori; Watanabe, Toshihiro; Mutoh, Shingo; Hasegawa, Kimiko; Kouguchi, Hirokazu; Sagane, Yoshimasa; Fujinaga, Yukako; Oguma, Keiji; Ohyama, Tohru

    2005-05-01

    The 650 kDa large toxin complex (L-TC) produced by Clostridium botulinum serotype D strain 4947 (D-4947) has a subunit structure composed of unnicked components, i.e. neurotoxin (NT), non-toxic non-haemagglutinin (NTNHA) and three haemagglutinin subcomponents (HA-70, HA-33 and HA-17). In this study, subunit interactions were investigated through the susceptibilities of the toxin components to limited trypsin proteolysis. Additionally, complex forms were reconstituted in vitro by various combinations of individual components. Trypsin treatment of intact D-4947 L-TC led to the formation of mature L-TC with nicks at specific sites of each component, which is usually observed in other strains of serotype D. NT, NTNHA and HA-17 were cleaved at their specific sites in either the single or complex forms, but HA-33 showed no sign of proteolysis. Unlike the other components, HA-70 was digested into random fragments as a single form, but it was cleaved into two fragments in the complex form. Based on the relative position of exposed or hidden regions of the individual components in the complex derived from their tryptic susceptibilities, an assembly model is proposed for the arrangement of individual subunits in the botulinum L-TC.

  10. Functional significance of conserved histidines and arginines in the 49-kDa subunit of mitochondrial complex I.

    PubMed

    Grgic, Ljuban; Zwicker, Klaus; Kashani-Poor, Noushin; Kerscher, Stefan; Brandt, Ulrich

    2004-05-14

    We have studied the ubiquinone-reducing catalytic core of NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica by a series of point mutations replacing conserved histidines and arginines in the 49-kDa subunit. Our results show that histidine 226 and arginine 141 probably do not ligate iron-sulfur cluster N2 but that exchanging these residues specifically influences the properties of this redox center. Histidines 91 and 95 were found to be essential for ubiquinone reductase activity of complex I. Mutations at the C-terminal arginine 466 affected ubiquinone affinity and inhibitor sensitivity but also destabilized complex I. These results provide further support for a high degree of structural conservation between the 49-kDa subunit of complex I and its ancestor, the large subunit of water-soluble [NiFe] hydrogenases. In several mutations of histidine 226, arginine 141, and arginine 466 the characteristic EPR signatures of iron-sulfur cluster N2 became undetectable, but specific, inhibitor-sensitive ubiquinone reductase activity was only moderately reduced. As we could not find spectroscopic indications for a modified cluster N2, we concluded that these complex I mutants were lacking most of this redox center but were still capable of catalyzing inhibitor-resistant ubiquinone reduction at near normal rates. We discuss that this at first surprising scenario may be explained by electron transfer theory; after removal of a single redox center in a chain, electron transfer rates are predicted to be still much faster than steady-state turnover of complex I. Our results question some of the central mechanistic functions that have been put forward for iron-sulfur cluster N2.

  11. Alteration of Skin Wound Healing in Keratinocyte-Specific Mediator Complex Subunit 1 Null Mice

    PubMed Central

    Inui, Shigeki; Reddy, Janardan K.; Itami, Satoshi

    2014-01-01

    MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1epi−/−) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1epi−/− and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1epi−/− mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1epi−/− mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1epi−/− keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1epi−/− keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1epi−/− keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1epi−/− keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1epi−/− mice. On the other hand, skin wound healing in 6-month-old Med1epi−/− mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1epi−/− mice, indicating a decreased

  12. Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing.

    PubMed

    Sommer, Natascha; Hüttemann, Maik; Pak, Oleg; Scheibe, Susan; Knoepp, Fenja; Sinkler, Christopher; Malczyk, Monika; Gierhardt, Mareike; Esfandiary, Azadeh; Kraut, Simone; Jonas, Felix; Veith, Christine; Aras, Siddhesh; Sydykov, Akylbek; Alebrahimdehkordi, Nasim; Giehl, Klaudia; Hecker, Matthias; Brandes, Ralf P; Seeger, Werner; Grimminger, Friedrich; Ghofrani, Hossein A; Schermuly, Ralph T; Grossman, Lawrence I; Weissmann, Norbert

    2017-08-04

    Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. Isolated ventilated and perfused lungs from Cox4i2(-/-) mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2(-/-) mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2(-/-) PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2(-/-) PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2(-/-) PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after

  13. Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes

    PubMed Central

    Sun, Weina; McCrory, Thomas S.; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell

    2014-01-01

    ABSTRACT Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998

  14. Fungal-specific subunits of the Candida albicans mitochondrial complex I drive diverse cell functions including cell wall synthesis.

    PubMed

    She, Xiaodong; Khamooshi, Kasra; Gao, Yin; Shen, Yongnian; Lv, Yuxia; Calderone, Richard; Fonzi, William; Liu, Weida; Li, Dongmei

    2015-09-01

    Our published research has focused on the role of Goa1p, an apparent regulator of the Candida albicans mitochondrial complex I (CI). Lack of Goa1p affects optimum cell growth, CI activity and virulence. Eukaryotic CI is composed of a core of 14 alpha-proteobacterial subunit proteins and a variable number of supernumerary subunit proteins. Of the latter group of proteins, one (NUZM) is fungal specific and the other (NUXM) is found in fungi, algae and plants, but is not a mammalian CI subunit protein. We have established that NUXM is orf19.6607 and NUZM is orf19.287 in C. albicans. Herein, we validate both subunit proteins as NADH:ubiquinone oxidoreductases (NUO) and annotate their gene functions. To accomplish these objectives, we compared null mutants of each with wild type (WT) and gene-reconstituted strains. Genetic mutants of genes NUO1 (orf19.6607) and NUO2 (orf19.287), not surprisingly, each had reduced oxygen consumption, decreased mitochondrial redox potential, decreased CI activity, increased reactive oxidant species (ROS) and decreased chronological ageing in vitro. Loss of either gene results in disassembly of CI. Transcriptional profiling of both mutants indicated significant down-regulation of genes of carbon metabolism, as well as up-regulation of mitochondrial-associated gene families that may occur to compensate for the loss of CI activity. Profiling of both mutants also demonstrated a loss of cell wall β-mannosylation but not in a conserved CI subunit (ndh51Δ). The profiling data may indicate specific functions driven by the enzymatic activity of Nuo1p and Nuo2p. Of importance, each mutant is also avirulent in a murine blood-borne, invasive model of candidiasis associated with their reduced colonization of tissues. Based on their fungal specificity and roles in virulence, we suggest both as drug targets for antifungal drug discovery.

  15. Fungal-specific subunits of the Candida albicans mitochondrial complex I drive diverse cell functions including cell wall synthesis

    PubMed Central

    She, Xiaodong; Khamooshi, Kasra; Gao, Yin; Shen, Yongnian; Lv, Yuxia; Calderone, Richard; Fonzi, William; Liu, Weida; Li, Dongmei

    2015-01-01

    Summary Our published research has focused upon the role of Goa1p, an apparent regulator of the Candida albicans mitochondrial complex I (CI). Lack of Goa1p effects optimum cell growth, CI activity, and virulence. Eukaryotic CI is composed of a core of 14 alpha-proteobacterial subunit proteins and a variable number of supernumerary subunit proteins. Of the latter group of proteins, one (NUZM) is fungal-specific, and a second (NUXM) is found in fungi, algae and plants but is not a mammalian CI subunit protein. We have established that NUXM is orf19.6607 and NUZM is orf19.287 in C. albicans. Herein, we validate both subunit proteins as NADH:ubiquinone oxidoreductases (NUO) and annotate their gene functions. To accomplish these objectives, we compared null mutants of each with WT and gene-reconstituted strains. Genetic mutants of genes NUO1 (19.6607) and NUO2 (19.287), not surprisingly, each had reduced oxygen consumption, decreased mitochondrial redox potential, decreased CI activity, increased reactive oxidant species (ROS), and a decrease in chronological aging in vitro. Loss of either gene results in a disassembly of CI. Transcriptional profiling of both mutants indicated significant down regulation of genes of carbon metabolism, as well as upregulation of mitochondrial-associated gene families which may occur to compensate for the loss of CI activity. Profiling of both mutants also demonstrated a loss of cell wall β-mannosylation but not in a conserved CI subunit (ndh51Δ). The profiling data may indicate specific functions driven by the enzymatic activity of Nuo1p and Nuo2p. Of importance, each mutant is also avirulent in a murine blood-borne, invasive model of candidiasis associated with their reduced colonization of tissues. Based upon their fungal-specificity and roles in virulence, we suggest both as drug targets for antifungal drug discovery. PMID:25801605

  16. Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.

    PubMed

    Wooddell, C I; Burgess, R R

    2000-11-07

    The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.

  17. Mitochondrial bioenergetics and redox state are unaltered in Trypanosoma cruzi isolates with compromised mitochondrial complex I subunit genes.

    PubMed

    Carranza, Julio César; Kowaltowski, Alicia J; Mendonça, Marco Aurélio G; de Oliveira, Thays C; Gadelha, Fernanda R; Zingales, Bianca

    2009-06-01

    In trypanosomatids the involvement of mitochondrial complex I in NADH oxidation has long been debated. Here, we took advantage of natural Trypanosoma cruzi mutants which present conspicuous deletions in ND4, ND5 and ND7 genes coding for complex I subunits to further investigate its functionality. Mitochondrial bioenergetics of wild type and complex I mutants showed no significant differences in oxygen consumption or respiratory control ratios in the presence of NADH-linked substrates or FADH(2)-generating succinate. No correlation could be established between mitochondrial membrane potentials and ND deletions. Since release of reactive oxygen species occurs at complex I, we measured mitochondrial H(2)O(2) formation induced by different substrates. Significant differences not associated to ND deletions were observed among the parasite isolates, demonstrating that these mutations are not important for the control of oxidant production. Our data support the notion that complex I has a limited function in T. cruzi.

  18. The changing of the guard: the Pto/Prf receptor complex of tomato and pathogen recognition.

    PubMed

    Ntoukakis, Vardis; Saur, Isabel M L; Conlan, Brendon; Rathjen, John P

    2014-08-01

    One important model for disease resistance is the Prf recognition complex of tomato, which responds to different bacterial effectors. Prf incorporates a protein kinase called Pto as its recognition domain that mimics effector virulence targets, and activates resistance after interaction with specific effectors. Recent findings show that this complex is oligomeric, and reveal how this impacts mechanism. Oligomerisation brings two or more kinases into proximity, where they can phosphorylate each other after effector perception. Effector attack on one kinase activates another in trans, constituting a molecular trap for the effector. Oligomerisation of plant resistance proteins may be a general concept that broadens pathogen recognition and restricts the ability of pathogens to evolve virulence.

  19. Recognition of complex human behaviours using 3D imaging for intelligent surveillance applications

    NASA Astrophysics Data System (ADS)

    Yao, Bo; Lepley, Jason J.; Peall, Robert; Butler, Michael; Hagras, Hani

    2016-10-01

    We introduce a system that exploits 3-D imaging technology as an enabler for the robust recognition of the human form. We combine this with pose and feature recognition capabilities from which we can recognise high-level human behaviours. We propose a hierarchical methodology for the recognition of complex human behaviours, based on the identification of a set of atomic behaviours, individual and sequential poses (e.g. standing, sitting, walking, drinking and eating) that provides a framework from which we adopt time-based machine learning techniques to recognise complex behaviour patterns.

  20. Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

    PubMed Central

    Huang, Da Wei; Fanti, Laura; Pak, Daniel T.S.; Botchan, Michael R.; Pimpinelli, Sergio; Kellum, Rebecca

    1998-01-01

    The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae. PMID:9679132

  1. Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA*

    PubMed Central

    Hoshina, Shoko; Yura, Kei; Teranishi, Honami; Kiyasu, Noriko; Tominaga, Ayumi; Kadoma, Haruka; Nakatsuka, Ayaka; Kunichika, Tomoko; Obuse, Chikashi; Waga, Shou

    2013-01-01

    Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs. PMID:24003239

  2. Human origin recognition complex binds preferentially to G-quadruplex-preferable RNA and single-stranded DNA.

    PubMed

    Hoshina, Shoko; Yura, Kei; Teranishi, Honami; Kiyasu, Noriko; Tominaga, Ayumi; Kadoma, Haruka; Nakatsuka, Ayaka; Kunichika, Tomoko; Obuse, Chikashi; Waga, Shou

    2013-10-18

    Origin recognition complex (ORC), consisting of six subunits ORC1-6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs.

  3. Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing.

    PubMed

    Shareef, M M; King, C; Damaj, M; Badagu, R; Huang, D W; Kellum, R

    2001-06-01

    Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

  4. Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

    PubMed Central

    Shareef, Mohammed Momin; King, Chadwick; Damaj, Mona; Badagu, RamaKrishna; Huang, Da Wei; Kellum, Rebecca

    2001-01-01

    Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit. PMID:11408576

  5. β-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Specificities; Physical Interaction with Nitrate Reductase Specifically Occurs via AKINβ1-Subunit1[C][OA

    PubMed Central

    Polge, Cécile; Jossier, Mathieu; Crozet, Pierre; Gissot, Lionel; Thomas, Martine

    2008-01-01

    The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one α-type catalytic subunit and two γ- and β-type regulatory subunits. In yeast it has been proposed that the β-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three β-type subunits of Arabidopsis (Arabidopsis thaliana; AKINβ1, AKINβ2, and AKINβ3) restore the growth phenotype of the yeast sip1Δsip2Δgal83Δ triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINβ promoter∷β-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the β-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each β-type subunit in plants. PMID:18768910

  6. Beta-subunits of the SnRK1 complexes share a common ancestral function together with expression and function specificities; physical interaction with nitrate reductase specifically occurs via AKINbeta1-subunit.

    PubMed

    Polge, Cécile; Jossier, Mathieu; Crozet, Pierre; Gissot, Lionel; Thomas, Martine

    2008-11-01

    The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one alpha-type catalytic subunit and two gamma- and beta-type regulatory subunits. In yeast it has been proposed that the beta-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three beta-type subunits of Arabidopsis (Arabidopsis thaliana; AKINbeta1, AKINbeta2, and AKINbeta3) restore the growth phenotype of the yeast sip1Deltasip2Deltagal83Delta triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINbeta promoterbeta-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the beta-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each beta-type subunit in plants.

  7. Crystallization of the Photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotianatabacum

    PubMed Central

    Piano, Dario; El Alaoui, Sabah; Korza, Henryk J.; Filipek, Renata; Sabala, Izabela; Haniewicz, Patrycja; Buechel, Claudia; De Sanctis, Daniele

    2010-01-01

    Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b559) was purified according to the protocol of Fey et al. (BBA 12:1501–1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group. PMID:21063907

  8. Subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ

    SciTech Connect

    Smith, R.A.; Branca, A.A.; Reichert, L.E. Jr.

    1985-11-15

    Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs.

  9. Effects of cocaine administration on receptor binding and subunits mRNA of GABA(A)-benzodiazepine receptor complexes.

    PubMed

    Suzuki, T; Abe, S; Yamaguchi, M; Baba, A; Hori, T; Shiraishi, H; Ito, T

    2000-11-01

    The effects of intermittent intraperitoneal (i.p.) administration of cocaine (20 mg/kg) on GABA(A)-benzodiazepine (BZD) receptors labeled by t-[(35)S]butylbicyclophosphorothionate (TBPS), and on several types of mRNA subunits were investigated in rat brain by in vitro quantitative receptor autoradiography and in situ hybridization. Phosphor screen imaging with high sensitivity and a wide linear range of response was utilized for imaging analysis. There was a significant decrease in the level of alpha 1, alpha 6, beta 2, beta 3, and gamma 2 subunits mRNA, with no alteration of [(35)S]TBPS binding in any regions in the brain of rats at 1 h following a single injection of cocaine. In chronically treated animals, the mean scores of stereotyped behavior were increased with the number of injections. The level of beta 3 subunit mRNA was decreased in the cortices and caudate putamen, at 24 h after a final injection of chronic administrations for 14 days. In the withdrawal from cocaine, the frontal cortex and hippocampal complexes showed a significant increase in [(35)S]TBPS binding and alpha1 and beta 3 subunit mRNA in the rats 1 week after a cessation of chronic administration of cocaine. These findings suggest that the disruption of GABA(A)-BZD receptor formation is closely involved in the development of cocaine-related behavioral disturbances. Further studies on the physiological functions on GABA(A)-BZD receptor complex will be necessary for an explanation of the precise mechanisms underlying the acute effects, development of hypersensitization, and withdrawal state of cocaine. Copyright 2000 Wiley-Liss, Inc.

  10. Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle.

    PubMed Central

    Nagley, P; Farrell, L B; Gearing, D P; Nero, D; Meltzer, S; Devenish, R J

    1988-01-01

    A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the F0/F1-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an N-terminal cleavable transit peptide (the leader sequence from Neurospora crassa ATPase subunit 9). The successful incorporation of the imported subunit 8 into functional ATPase complexes after transformation with pLF1 expressing N9/Y8 was indicated by the efficient genetic complementation of respiratory growth defects of aap1 mit- mutants, which lack endogenous subunit 8. The reconstitution of ATPase function was confirmed by biochemical assays of ATPase performance in mitochondria and by immunochemical analyses that demonstrated the assembly of the cytoplasmically synthesized subunit 8 into the ATPase complex. Reconstitution of ATPase function required the cytoplasmically synthesized subunit to have a transit peptide. The strategy for importation and reconstitution developed for subunit 8 leads to a systematic approach to the directed manipulation of mitochondrially encoded membrane-associated proteins that has general implications for exploring membrane biogenesis mechanistically and evolutionarily. Images PMID:2895470

  11. Mass Spectrometry Reveals Differences in Stability and Subunit Interactions between Activated and Nonactivated Conformers of the (αβγδ)4 Phosphorylase Kinase Complex*

    PubMed Central

    Lane, Laura A.; Nadeau, Owen W.; Carlson, Gerald M.; Robinson, Carol V.

    2012-01-01

    Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, β, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the β subunit, the predominant subunit responsible for PhK's activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple β subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αβγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall

  12. Crystal Structure in the Vivo-Assembled Bacillus subtilis Spx/RNA Polymerase alpha Subunit C-Terminal Domain Complex

    SciTech Connect

    Lamour, V.; Westblade, L; Campbell, E; Darst, S

    2009-01-01

    The Bacillus subtilis Spx protein is a global transcription factor that interacts with the C-terminal domain of the RNA polymerase {alpha} subunit ({alpha}CTD) and regulates transcription of genes involved in thiol-oxidative stress, sporulation, competence, and organosulfur metabolism. Here we determined the X-ray crystal structure of the Spx/{alpha}CTD complex from an entirely new crystal form than previously reported [Newberry, K.J., Nakano, S., Zuber, P., Brennan, R.G., 2005. Crystal structure of the Bacillus subtilis anti-alpha, global transcriptional regulator, Spx, in complex with the alpha C-terminal domain of RNA polymerase. Proc. Natl. Acad. Sci. USA 102, 15839-15844]. Comparison of the previously reported sulfate-bound complex and our sulfate-free complex reveals subtle conformational changes that may be important for the role of Spx in regulating organosulfur metabolism.

  13. Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex

    PubMed Central

    Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Williams, Carole; Miller, Christopher

    2016-01-01

    Mitochondrial Ca2+ uptake, a process crucial for bioenergetics and Ca2+ signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca2+-activated Ca2+ channel, with the Ca2+ pore formed by the MCU protein and Ca2+-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca2+ permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca2+ landscape. DOI: http://dx.doi.org/10.7554/eLife.15545.001 PMID:27099988

  14. Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes.

    PubMed

    Nicoletti; Witt; Ludwig; Brunori; Malatesta

    1998-07-20

    Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I-IV is obtained when the purification procedure is performed in beta-d-dodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I-II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.

  15. TRIM5α associates with proteasomal subunits in cells while in complex with HIV-1 virions

    PubMed Central

    2011-01-01

    Background The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues. Results The only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions. Conclusions Taken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction. PMID:22078707

  16. Emotion recognition deficits among persons with schizophrenia: Beyond stimulus complexity level and presentation modality.

    PubMed

    Feingold, Daniel; Hasson-Ohayon, Ilanit; Laukka, Petri; Vishne, Tali; Dembinsky, Yael; Kravets, Shlomo

    2016-06-30

    Studies have shown that persons with schizophrenia have lower accuracy in emotion recognition compared to persons without schizophrenia. However, the impact of the complexity level of the stimuli or the modality of presentation has not been extensively addressed. Forty three persons with a diagnosis of schizophrenia and 43 healthy controls, matched for age and gender, were administered tests assessing emotion recognition from stimuli with low and high levels of complexity presented via visual, auditory and semantic channels. For both groups, recognition rates were higher for high-complexity stimuli compared to low-complexity stimuli. Additionally, both groups obtained higher recognition rates for visual and semantic stimuli than for auditory stimuli, but persons with schizophrenia obtained lower accuracy than persons in the control group for all presentation modalities. Persons diagnosed with schizophrenia did not present a level of complexity specific deficit or modality-specific deficit compared to healthy controls. Results suggest that emotion recognition deficits in schizophrenia are beyond level of complexity of stimuli and modality, and present a global difficulty in cognitive functioning.

  17. A Specific Set of Exon Junction Complex Subunits Is Required for the Nuclear Retention of Unspliced RNAs in Caenorhabditis elegans

    PubMed Central

    Shiimori, Masami; Inoue, Kunio

    2013-01-01

    The exon junction complex (EJC) is highly conserved in many organisms and is involved in various steps of mRNA metabolism. During the course of investigating the role of EJC in the germ line sex determination of the nematode Caenorhabditis elegans, we found that depletion of one of the three core subunits (Y14, MAG-1, and eukaryotic translation initiation factor 4III [eIF4AIII]) or one auxiliary subunit (UAP56) of EJC resulted in the cytoplasmic leakage of unspliced RNAs from almost all of the C. elegans protein-coding genes examined thus far. This leakage was also observed with the depletion of several splicing factors, including SF3b, IBP160, and PRP19, all of which genetically interacted with Y14. We also found that Y14 physically interacts with both pre-mRNA and spliceosomal U snRNAs, especially U2 snRNA, and that the interaction was abolished when both IBP160 and PRP19 were depleted. Our results strongly suggest that a specific set of EJC subunits is recruited onto introns and interacts with components of the spliceosome, including U2 snRNP, to provide a critical signal for the surveillance and nuclear retention of unspliced RNAs in C. elegans. PMID:23149939

  18. Crystal structures of ricin toxin's enzymatic subunit (RTA) in complex with neutralizing and non-neutralizing single-chain antibodies.

    PubMed

    Rudolph, Michael J; Vance, David J; Cheung, Jonah; Franklin, Matthew C; Burshteyn, Fiana; Cassidy, Michael S; Gary, Ebony N; Herrera, Cristina; Shoemaker, Charles B; Mantis, Nicholas J

    2014-08-26

    Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.

  19. Inducible Repression of Nuclear-Encoded Subunits of the Cytochrome b6f Complex in Tobacco Reveals an Extraordinarily Long Lifetime of the Complex1[W][OPEN

    PubMed Central

    Hojka, Marta; Thiele, Wolfram; Tóth, Szilvia Z.; Lein, Wolfgang; Bock, Ralph; Schöttler, Mark Aurel

    2014-01-01

    The biogenesis of the cytochrome b6f complex in tobacco (Nicotiana tabacum) seems to be restricted to young leaves, suggesting a high lifetime of the complex. To directly determine its lifetime, we employed an ethanol-inducible RNA interference (RNAi) approach targeted against the essential nuclear-encoded Rieske protein (PetC) and the small M subunit (PetM), whose function in higher plants is unknown. Young expanding leaves of both PetM and PetC RNAi transformants bleached rapidly and developed necroses, while mature leaves, whose photosynthetic apparatus was fully assembled before RNAi induction, stayed green. In line with these phenotypes, cytochrome b6f complex accumulation and linear electron transport capacity were strongly repressed in young leaves of both RNAi transformants, showing that the M subunit is as essential for cytochrome b6f complex accumulation as the Rieske protein. In mature leaves, all photosynthetic parameters were indistinguishable from the wild type even after 14 d of induction. As RNAi repression of PetM and PetC was highly efficient in both young and mature leaves, these data indicate a lifetime of the cytochrome b6f complex of at least 1 week. The switch-off of cytochrome b6f complex biogenesis in mature leaves may represent part of the first dedicated step of the leaf senescence program. PMID:24963068

  20. Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue.

    PubMed

    Richard, Allison J; Hang, Hardy; Stephens, Jacqueline M

    2017-10-05

    STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Since STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene cish (cytokine inducible SH2 containing protein). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  1. Diazirine photocrosslinking recruits activated FTO demethylase complexes for specific N(6)-methyladenosine recognition.

    PubMed

    Jeong, Hyun Seok; Hayashi, Gosuke; Okamoto, Akimitsu

    2015-06-19

    N(6)-methyladenosine (m(6)A) is a prevalent modification of RNAs. m(6)A exists in mRNA and plays an important role in RNA biological pathways and in RNA epigenetic regulation. We applied diazirine photocrosslinking to the event of m(6)A recognition mediated by the fat mass and obesity associated (FTO) demethylase. A highly photoreactive diazirine adjacent to m(6)A on the RNA successfully recruited activated FTO complexes with an m(6)A preference. The process of recognition of m(6)A via FTO using diazirine photocrosslinking was controlled by the α-ketoglutarate (α-KG) cosubstrate and the Fe(II) cofactor, which are involved in m(6)A oxidative demethylation. In addition, FTO bound to ssRNAs prior to the m(6)A recognition process. Diazirine photocrosslinking contributes to increasing the chances of capturing activated FTO complexes with specific m(6)A recognition and provides new insights into the dynamic FTO oxidative demethylation process.

  2. Molecular Recognition in the Oxidation of Catechols by Dicobalt-BISDIEN Dioxygen Complexes

    DTIC Science & Technology

    1992-01-30

    Recognition in the Oxidation of Catechols by Dicobalt-RISDIEN Dioxygen Complexes Lizete F S Cezar and Bruno Szpoganicz Departamento de Quimica ...bridged bi- nuclear Co(II)-BISDIEN dioxygen complexes; Co20 2 LCat2 + is the bivalent form, and Co20 2 (OH)LCat + and Co 20 2 (OH)2 Cat° are hydroxo

  3. Characterization of the RnfB and RnfG Subunits of the Rnf Complex from the Archaeon Methanosarcina acetivorans

    PubMed Central

    Suharti, Suharti; Wang, Mingyu; de Vries, Simon; Ferry, James G.

    2014-01-01

    Rnf complexes are redox-driven ion pumps identified in diverse species from the domains Bacteria and Archaea, biochemical characterizations of which are reported for two species from the domain Bacteria. Here, we present characterizations of the redox-active subunits RnfG and RnfB from the Rnf complex of Methanosarcina acetivorans, an acetate-utilizing methane-producing species from the domain Archaea. The purified RnfG subunit produced in Escherichia coli fluoresced in SDS-PAGE gels under UV illumination and showed a UV-visible spectrum typical of flavoproteins. The Thr166Gly variant of RnfG was colorless and failed to fluoresce under UV illumination confirming a role for Thr166 in binding FMN. Redox titration of holo-RnfG revealed a midpoint potential of −129 mV for FMN with n = 2. The overproduced RnfG was primarily localized to the membrane of E. coli and the sequence contained a transmembrane helix. A topological analysis combining reporter protein fusion and computer predictions indicated that the C-terminal domain containing FMN is located on the outer aspect of the cytoplasmic membrane. The purified RnfB subunit produced in E. coli showed a UV-visible spectrum typical of iron-sulfur proteins. The EPR spectra of reduced RnfB featured a broad spectral shape with g values (2.06, 1.94, 1.90, 1.88) characteristic of magnetically coupled 3Fe-4S and 4Fe-4S clusters in close agreement with the iron and acid-labile sulfur content. The ferredoxin specific to the aceticlastic pathway served as an electron donor to RnfB suggesting this subunit is the entry point of electrons to the Rnf complex. The results advance an understanding of the organization and biochemical properties of the Rnf complex and lay a foundation for further understanding the overall mechanism in the pathway of methane formation from acetate. PMID:24836163

  4. The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance, but Not Sister Chromatid Cohesion, during Drosophila Female Meiosis

    PubMed Central

    Lehner, Christian F.; Heidmann, Stefan K.

    2014-01-01

    Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin. PMID:25101996

  5. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    PubMed

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. The cohesin subunit Rad21 is required for synaptonemal complex maintenance, but not sister chromatid cohesion, during Drosophila female meiosis.

    PubMed

    Urban, Evelin; Nagarkar-Jaiswal, Sonal; Lehner, Christian F; Heidmann, Stefan K

    2014-08-01

    Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin.

  7. Conservation of core complex subunits shaped the structure and function of photosystem I in the secondary endosymbiont alga Nannochloropsis gaditana.

    PubMed

    Alboresi, Alessandro; Le Quiniou, Clotilde; Yadav, Sathish K N; Scholz, Martin; Meneghesso, Andrea; Gerotto, Caterina; Simionato, Diana; Hippler, Michael; Boekema, Egbert J; Croce, Roberta; Morosinotto, Tomas

    2017-01-01

    Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained.

  8. Recognition of preproteins by the isolated TOM complex of mitochondria.

    PubMed

    Stan, T; Ahting, U; Dembowski, M; Künkele, K P; Nussberger, S; Neupert, W; Rapaport, D

    2000-09-15

    A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear-encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound efficiently in a specific manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using fluorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The affinity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease-treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

  9. The Pex1/Pex6 complex is a heterohexameric AAA+ motor with alternating and highly coordinated subunits.

    PubMed

    Gardner, Brooke M; Chowdhury, Saikat; Lander, Gabriel C; Martin, Andreas

    2015-03-27

    Pex1 and Pex6 are Type-2 AAA+ ATPases required for the de novo biogenesis of peroxisomes. Mutations in Pex1 and Pex6 account for the majority of the most severe forms of peroxisome biogenesis disorders in humans. Here, we show that the ATP-dependent complex of Pex1 and Pex6 from Saccharomyces cerevisiae is a heterohexamer with alternating subunits. Within the Pex1/Pex6 complex, only the D2 ATPase ring hydrolyzes ATP, while nucleotide binding in the D1 ring promotes complex assembly. ATP hydrolysis by Pex1 is highly coordinated with that of Pex6. Furthermore, Pex15, the membrane anchor required for Pex1/Pex6 recruitment to peroxisomes, inhibits the ATP-hydrolysis activity of Pex1/Pex6.

  10. Luminescent complexes of terbium ion for molecular recognition of ibuprofen.

    PubMed

    Selivanova, Natalia; Vasilieva, Kristina; Galyametdinov, Yury

    2014-05-01

    The complexation behavior and luminescent properties of terbium (Tb(3+) ) complexes containing bi-dental ligands were studied: nitrogen - 1,10-phenanthroline, and oxygen - trifluoroacetylacetone as well as acetylacetone ligands with ibuprofen (Ibu; a non-steroidal anti-inflammatory drug). Aqueous and aqueous alcohol microheterogeneous solutions were used as media. The effects of solubilization by various micellar solutions, pH and ligand type on luminescent properties of Tb(3+) complexes were investigated. Sensitized luminescence of mixed ligand complex Tb(1,10-phenanthroline)-Ibu and dynamic quenching effect in complex Tb(trifluoroacetylacetone)3 -Ibu allow Ibu determination with the limit of detection 5.3 × 10(-8)  mol/L and 1.26 × 10(-6)  mol/L, respectively.

  11. The crystal structure of the human nascent polypeptide-associated complex domain reveals a nucleic acid-binding region on the NACA subunit .

    PubMed

    Liu, Yiwei; Hu, Yingxia; Li, Xu; Niu, Liwen; Teng, Maikun

    2010-04-06

    In archaea and eukaryotes, the nascent polypeptide-associated complex (NAC) is one of the cytosolic chaperones that contact the nascent polypeptide chains as they emerge from the ribosome and assist in post-translational processes. The eukaryotic NAC is a heterodimer, and its two subunits form a stable complex through a dimerizing domain called the NAC domain. In addition to acting as a protein translation chaperone, the NAC subunits also function individually in transcriptional regulation. Here we report the crystal structure of the human NAC domain, which reveals the manner of human NAC dimerization. On the basis of the structure, we identified a region in the NAC domain of the human NAC alpha-subunit as a new nucleic acid-binding region, which is blocked from binding nucleic acids in the heterodimeric complex by a helix region in the beta-subunit.

  12. Mitochondrial hepato-encephalopathy due to deficiency of QIL1/MIC13 (C19orf70), a MICOS complex subunit.

    PubMed

    Zeharia, Avraham; Friedman, Jonathan R; Tobar, Ana; Saada, Ann; Konen, Osnat; Fellig, Yacov; Shaag, Avraham; Nunnari, Jodi; Elpeleg, Orly

    2016-12-01

    The mitochondrial inner membrane possesses distinct subdomains including cristae, which are lamellar structures invaginated into the mitochondrial matrix and contain the respiratory complexes. Generation of inner membrane domains requires the complex interplay between the respiratory complexes, mitochondrial lipids and the recently identified mitochondrial contact site and cristae organizing system (MICOS) complex. Proper organization of the mitochondrial inner membrane has recently been shown to be important for respiratory function in yeast. Here we aimed at a molecular diagnosis in a brother and sister from a consanguineous family who presented with a neurodegenerative disorder accompanied by hyperlactatemia, 3-methylglutaconic aciduria, disturbed hepatocellular function with abnormal cristae morphology in liver and cerebellar and vermis atrophy, which suggest mitochondrial dysfunction. Using homozygosity mapping and exome sequencing the patients were found to be homozygous for the p.(Gly15Glufs*75) variant in the QIL1/MIC13 (C19orf70) gene. QIL1/MIC13 is a constituent of MICOS, a six subunit complex that helps to form and/or stabilize cristae junctions and determine the placement, distribution and number of cristae within mitochondria. In patient fibroblasts both MICOS subunits QIL1/MIC13 and MIC10 were absent whereas MIC60 was present in a comparable abundance to that of the control. We conclude that QIL1/MIC13 deficiency in human, is associated with disassembly of the MICOS complex, with the associated aberration of cristae morphology and mitochondrial respiratory dysfunction. 3-Methylglutaconic aciduria is associated with variants in genes encoding mitochondrial inner membrane organizing determinants, including TAZ, DNAJC19, SERAC1 and QIL1/MIC13.

  13. Collision-Induced Release, Ion Mobility Separation, and Amino Acid Sequence Analysis of Subunits from Mass-Selected Noncovalent Protein Complexes

    NASA Astrophysics Data System (ADS)

    Rathore, Deepali; Dodds, Eric D.

    2014-09-01

    In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein-protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.

  14. Collision-induced release, ion mobility separation, and amino acid sequence analysis of subunits from mass-selected noncovalent protein complexes.

    PubMed

    Rathore, Deepali; Dodds, Eric D

    2014-09-01

    In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein-protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.

  15. Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

    SciTech Connect

    Nakai, Yuji; Shiratsuchi, Akiko; Manaka, Junko; Nakayama, Hiroshi; Takio, Koji; Zhang Jianting; Suganuma, Tatsuo; Nakanishi, Yoshinobu . E-mail: nakanaka@kenroku.kanazawa-u.ac.jp

    2005-09-10

    We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis.

  16. Genetic dissection of the budding yeast Arp2/3 complex: A comparison of the in vivo and structural roles of individual subunits

    PubMed Central

    Winter, Dirk C.; Choe, Elizabeth Y.; Li, Rong

    1999-01-01

    In previous work, we identified the yeast Arp2/3 complex, which localizes to cortical actin patches and is required for their motility and integrity in vivo. This complex contains proteins homologous to each subunit of the Acanthamoeba and human Arp2/3 complex except for a 40-kDa subunit (p40), which was missing from the purified yeast complex. Here, we demonstrate by using immunoprecipitation and gel-filtration analysis that Arc40p, the homolog of p40 identified from the yeast genome database, associates with the yeast Arp2/3 complex. We have carried out gene disruptions of each subunit of the yeast Arp2/3 complex to study each subunit’s role in the function of the complex. Surprisingly, we find that only ARC40 is fully essential for cell viability. Strains lacking each of the other subunits exhibit varying degrees of defects in cell growth and viability and in assembly and polarization of cortical actin patches. We have also examined each subunit’s role in maintaining the structural integrity of the Arp2/3 complex. Arp2p, Arp3p, and Arc40p fall into the monomer pool in Δarc19 and Δarc35 cells, suggesting that Arc19p and Arc35p are the central scaffolding components of the complex. Arp2p and Arp3p do not have major roles in maintaining complex integrity, and Arc15p is required for association of Arp2p and Arc40p, but not other subunits, with the complex. These results provide evidence that each subunit contributes differently to the assembly and function of the Arp2/3 complex. PMID:10377407

  17. Intracellular Targeting Signals and Lipid Specificity Determinants of the ALA/ALIS P4-ATPase Complex Reside in the Catalytic ALA α-Subunit

    PubMed Central

    Poulsen, Lisbeth R.; Hanisch, Susanne; Meffert, Katharina; Buch-Pedersen, Morten J.; Jakobsen, Mia K.; Pomorski, Thomas Günther; Palmgren, Michael G.

    2010-01-01

    Members of the P4 subfamily of P-type ATPases are believed to catalyze flipping of phospholipids across cellular membranes, in this way contributing to vesicle biogenesis in the secretory and endocytic pathways. P4-ATPases form heteromeric complexes with Cdc50-like proteins, and it has been suggested that these act as β-subunits in the P4-ATPase transport machinery. In this work, we investigated the role of Cdc50-like β-subunits of P4-ATPases for targeting and function of P4-ATPase catalytic α-subunits. We show that the Arabidopsis P4-ATPases ALA2 and ALA3 gain functionality when coexpressed with any of three different ALIS Cdc50-like β-subunits. However, the final cellular destination of P4-ATPases as well as their lipid substrate specificity are independent of the nature of the ALIS β-subunit they were allowed to interact with. PMID:20053675

  18. Essential structural and functional roles of the Cmr4 subunit in RNA cleavage by the Cmr CRISPR-Cas complex.

    PubMed

    Ramia, Nancy F; Spilman, Michael; Tang, Li; Shao, Yaming; Elmore, Joshua; Hale, Caryn; Cocozaki, Alexis; Bhattacharya, Nilakshee; Terns, Rebecca M; Terns, Michael P; Li, Hong; Stagg, Scott M

    2014-12-11

    The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

    PubMed Central

    Ohashi, Yohei; Soler, Nicolas; García Ortegón, Miguel; Zhang, Lufei; Kirsten, Marie L.; Perisic, Olga; Masson, Glenn R.; Burke, John E.; Jakobi, Arjen J.; Apostolakis, Apostolos A.; Johnson, Christopher M.; Ohashi, Maki; Ktistakis, Nicholas T.; Sachse, Carsten; Williams, Roger L.

    2016-01-01

    ABSTRACT The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies. PMID:27630019

  20. Identification and Characterization of Sa/Scc3p Subunits in the Xenopus and Human Cohesin Complexes

    PubMed Central

    Losada, Ana; Yokochi, Tomoki; Kobayashi, Ryuji; Hirano, Tatsuya

    2000-01-01

    A multisubunit protein complex, termed cohesin, plays an essential role in sister chromatid cohesion in yeast and in Xenopus laevis cell-free extracts. We report here that two distinct cohesin complexes exist in Xenopus egg extracts. A 14S complex (x-cohesinSA1) contains XSMC1, XSMC3, XRAD21, and a newly identified subunit, XSA1. In a second 12.5S complex (x-cohesinSA2), XSMC1, XSMC3, and XRAD21 associate with a different subunit, XSA2. Both XSA1 and XSA2 belong to the SA family of mammalian proteins and exhibit similarity to Scc3p, a recently identified component of yeast cohesin. In Xenopus egg extracts, x-cohesinSA1 is predominant, whereas x-cohesinSA2 constitutes only a very minor population. Human cells have a similar pair of cohesin complexes, but the SA2-type is the dominant form in somatic tissue culture cells. Immunolocalization experiments suggest that chromatin association of cohesinSA1 and cohesinSA2 may be differentially regulated. Dissociation of x-cohesinSA1 from chromatin correlates with phosphorylation of XSA1 in the cell-free extracts. Purified cdc2-cyclin B can phosphorylate XSA1 in vitro and reduce the ability of x-cohesinSA1 to bind to DNA or chromatin. These results shed light on the mechanism by which sister chromatid cohesion is partially dissolved in early mitosis, far before the onset of anaphase, in vertebrate cells. PMID:10931856

  1. In Vivo Identification of Photosystem II Light Harvesting Complexes Interacting with PHOTOSYSTEM II SUBUNIT S1[OPEN

    PubMed Central

    Franchin, Cinzia; Arrigoni, Giorgio

    2015-01-01

    Light is the primary energy source for photosynthetic organisms, but in excess, it can generate reactive oxygen species and lead to cell damage. Plants evolved multiple mechanisms to modulate light use efficiency depending on illumination intensity to thrive in a highly dynamic natural environment. One of the main mechanisms for protection from intense illumination is the dissipation of excess excitation energy as heat, a process called nonphotochemical quenching. In plants, nonphotochemical quenching induction depends on the generation of a pH gradient across thylakoid membranes and on the presence of a protein called PHOTOSYSTEM II SUBUNIT S (PSBS). Here, we generated Physcomitrella patens lines expressing histidine-tagged PSBS that were exploited to purify the native protein by affinity chromatography. The mild conditions used in the purification allowed copurifying PSBS with its interactors, which were identified by mass spectrometry analysis to be mainly photosystem II antenna proteins, such as LIGHT-HARVESTING COMPLEX B (LHCB). PSBS interaction with other proteins appears to be promiscuous and not exclusive, although the major proteins copurified with PSBS were components of the LHCII trimers (LHCB3 and LHCBM). These results provide evidence of a physical interaction between specific photosystem II light-harvesting complexes and PSBS in the thylakoids, suggesting that these subunits are major players in heat dissipation of excess energy. PMID:26069151

  2. Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    PubMed Central

    Génier, Samuel; Degrandmaison, Jade; Moreau, Pierrick; Labrecque, Pascale; Hébert, Terence E.; Parent, Jean-Luc

    2016-01-01

    Mechanisms that prevent aggregation and promote folding of nascent G protein–coupled receptors (GPCRs) remain poorly understood. We identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the β-isoform of thromboxane A2 receptor (TPβ) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPβ and β2-adrenergic receptor (β2AR) in HEK 293 cells, but also with endogenous β2AR. CCT7 depletion by small interfering RNA reduced total and cell-surface expression of both receptors and caused redistribution of the receptors to juxtanuclear aggresomes, significantly more so for TPβ than β2AR. Interestingly, Hsp90 coimmunoprecipitated with β2AR but virtually not with TPβ, indicating that nascent GPCRs can adopt alternative folding pathways. In vitro pull-down assays showed that both receptors can interact directly with CCT7 through their third intracellular loops and C-termini. We demonstrate that Trp334 in the TPβ C-terminus is critical for the CCT7 interaction and plays an important role in TPβ maturation and cell-surface expression. Of note, introducing a tryptophan in the corresponding position of the TPα isoform confers the CCT7-binding and maturation properties of TPβ. We show that an interaction with a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex is involved in regulating aggregation of nascent GPCRs and in promoting their proper maturation and expression. PMID:27708139

  3. Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.

    PubMed

    Dovega, Rebecca; Tsutakawa, Susan; Quistgaard, Esben M; Anandapadamanaban, Madhanagopal; Löw, Christian; Nordlund, Pär

    2014-01-01

    Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.

  4. Structural and Biochemical Characterization of Human PR70 in Isolation and in Complex with the Scaffolding Subunit of Protein Phosphatase 2A

    PubMed Central

    Dovega, Rebecca; Tsutakawa, Susan; Quistgaard, Esben M.; Anandapadamanaban, Madhanagopal; Löw, Christian; Nordlund, Pär

    2014-01-01

    Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B’’/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B’’/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B’’/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B’’ containing holoenzymes. PMID:25007185

  5. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    SciTech Connect

    Bhaskar,; Kumari, Neeti; Goyal, Neena

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  6. A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex.

    PubMed

    Dunn, Cory D; Lee, Marina S; Spencer, Forrest A; Jensen, Robert E

    2006-01-01

    Unlike many other organisms, the yeast Saccharomyces cerevisiae can tolerate the loss of mitochondrial DNA (mtDNA). Although a few proteins have been identified that are required for yeast cell viability without mtDNA, the mechanism of mtDNA-independent growth is not completely understood. To probe the relationship between the mitochondrial genome and cell viability, we conducted a microarray-based, genomewide screen for mitochondrial DNA-dependent yeast mutants. Among the several genes that we discovered is MGR1, which encodes a novel subunit of the i-AAA protease complex located in the mitochondrial inner membrane. mgr1Delta mutants retain some i-AAA protease activity, yet mitochondria lacking Mgr1p contain a misassembled i-AAA protease and are defective for turnover of mitochondrial inner membrane proteins. Our results highlight the importance of the i-AAA complex and proteolysis at the inner membrane in cells lacking mitochondrial DNA.

  7. Does aging impair first impression accuracy? Differentiating emotion recognition from complex social inferences.

    PubMed

    Krendl, Anne C; Rule, Nicholas O; Ambady, Nalini

    2014-09-01

    Young adults can be surprisingly accurate at making inferences about people from their faces. Although these first impressions have important consequences for both the perceiver and the target, it remains an open question whether first impression accuracy is preserved with age. Specifically, could age differences in impressions toward others stem from age-related deficits in accurately detecting complex social cues? Research on aging and impression formation suggests that young and older adults show relative consensus in their first impressions, but it is unknown whether they differ in accuracy. It has been widely shown that aging disrupts emotion recognition accuracy, and that these impairments may predict deficits in other social judgments, such as detecting deceit. However, it is unclear whether general impression formation accuracy (e.g., emotion recognition accuracy, detecting complex social cues) relies on similar or distinct mechanisms. It is important to examine this question to evaluate how, if at all, aging might affect overall accuracy. Here, we examined whether aging impaired first impression accuracy in predicting real-world outcomes and categorizing social group membership. Specifically, we studied whether emotion recognition accuracy and age-related cognitive decline (which has been implicated in exacerbating deficits in emotion recognition) predict first impression accuracy. Our results revealed that emotion recognition accuracy did not predict first impression accuracy, nor did age-related cognitive decline impair it. These findings suggest that domains of social perception outside of emotion recognition may rely on mechanisms that are relatively unimpaired by aging.

  8. On the recognition of complex structures: Computer software using artificial intelligence applied to pattern recognition

    NASA Technical Reports Server (NTRS)

    Yakimovsky, Y.

    1974-01-01

    An approach to simultaneous interpretation of objects in complex structures so as to maximize a combined utility function is presented. Results of the application of a computer software system to assign meaning to regions in a segmented image based on the principles described in this paper and on a special interactive sequential classification learning system, which is referenced, are demonstrated.

  9. Thymoproteasome subunit-β5T generates peptide-MHC complexes specialized for positive selection

    PubMed Central

    Xing, Yan; Jameson, Stephen C.; Hogquist, Kristin A.

    2013-01-01

    Cortical thymic epithelial cells (cTECs) express a unique thymoproteasome subunit-β5T that plays an essential role in the development of CD8 T cells. In contrast, the immunoproteasome subunit-β5i is expressed in other thymic antigen-presenting cells (APCs). The thymoproteasome may generate peptides that are specialized for positive selection, or it may simply serve to generate peptides that are distinct from other APCs that cause negative selection, thereby promoting an overall larger number of surviving clones to mature and function in the immune system. To distinguish these models, we genetically engineered mice to express distinct peptide repertoires in cTECs vs. other APCs without expressing β5T, by generating β5t5i knockin mice, in which β5i replaced β5T in cTECs. When such animals were crossed to β5i−/− mice, β5i was exclusively expressed in cTECs, whereas β5 was expressed in other cells. However, this mouse did not support normal positive selection, suggesting that β5T generates peptides that are intrinsically better for positive selection (i.e., β5i could not replace β5T) and not merely because these peptides are distinct from peptides presented by other APCs. Finally, using an Nur77GFP reporter, we show that the T cells generated in the absence of β5T have higher reactivity to self, generating predominantly CD44hi memory phenotype peripheral CD8+ T cells. Altogether, our results suggest that the thymoproteasome supports positive selection by generating peptides that are optimized for the selection of weakly self-reactive, naïve T-cell clones. PMID:23569244

  10. Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of Trypanosoma cruzi.

    PubMed

    Chiurillo, Miguel A; Lander, Noelia; Bertolini, Mayara S; Storey, Melissa; Vercesi, Anibal E; Docampo, Roberto

    2017-05-09

    Trypanosoma cruzi is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of TcMCU, or its paralog TcMCUb, by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca(2+) uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca(2+) While TcMCU-knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of TcMCUb resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of TcMCUb, in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU.IMPORTANCE The finding of a mitochondrial calcium uniporter (MCU) in Trypanosoma cruzi was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for T. cruzi to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU. Copyright © 2017 Chiurillo et al.

  11. Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I.

    PubMed

    Sarkar, Sourav; Shenoy, Rajesh T; Dalgaard, Jacob Z; Newnham, Louise; Hoffmann, Eva; Millar, Jonathan B A; Arumugam, Prakash

    2013-01-01

    Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.

  12. Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II

    PubMed Central

    Foulquier, François; Vasile, Eliza; Schollen, Els; Callewaert, Nico; Raemaekers, Tim; Quelhas, Dulce; Jaeken, Jaak; Mills, Philippa; Winchester, Bryan; Krieger, Monty; Annaert, Wim; Matthijs, Gert

    2006-01-01

    The conserved oligomeric Golgi (COG) complex is a heterooctameric complex that regulates intraGolgi trafficking and the integrity of the Golgi compartment in eukaryotic cells. Here, we describe a patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex. This patient has a defect in both N- and O-glycosylation. Mass spectrometric analysis of the structures of the N-linked glycans released from glycoproteins from the patient's serum revealed a reduction in sialic acid and galactose residues. Peanut agglutinin (PNA) lectin staining revealed a decrease in sialic acids on core 1 mucin type O-glycans, indicating a combined defect in N- and O-glycosylation. Sequence analysis of the COG1 cDNA and gene identified a homozygous insertion of a single nucleotide (2659–2660insC), which is predicted to lead to a premature translation stop and truncation of the C terminus of the Cog1 protein by 80 amino acids. This mutation destabilizes several other COG subunits and alters their subcellular localization and hence the overall integrity of the COG complex. This results in reduced levels and/or altered Golgi localization of α-mannosidase II and β-1,4 galactosyltransferase I, which links it to the glycosylation deficiency. Transfection of primary fibroblasts of this patient with the full length hemagglutinin-tagged Cog1 indeed restored β-1,4 galactosyltransferase Golgi localization. We propose naming this disorder CDG-II/Cog1, or CDG-II caused by Cog1 deficiency. PMID:16537452

  13. Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome

    PubMed Central

    Vorobyeva, Nadezhda E.; Mazina, Marina U.; Golovnin, Anton K.; Kopytova, Daria V.; Gurskiy, Dmitriy Y.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Georgiev, Pavel G.; Krasnov, Aleksey N.

    2013-01-01

    Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein. PMID:23609538

  14. Visual recognition of complex medical lesions using 2D shape

    NASA Astrophysics Data System (ADS)

    Chodorowski, Artur; Gustavsson, Tomas; Mattsson, Ulf

    2000-06-01

    Different shape representation and classification methods for complex medical lesions were compared using oral lesions as a case study. The problem studied was the discrimination between potentially cancerous lesions, called leukoplakia, and other usually harmless lesions, called lichenoid reactions, which can appear in human oral cavities. The classification problem is difficult because these lesions vary in shape within classes and there are no easily recognizable characteristics. The representations evaluated were the centroidal profile function, the curvature function, and polar and complex coordinate functions. From these representations, translation, scale and rotation independent features were derived using Fourier transformations, auto-regressive modeling, and Zernike moments. A nonparametric kNN classifier with the leave-one-out cross-validation method was used as a classifier. An overall classification accuracy of about 84% was achieved using only the shape properties of the lesions, compared with a human visual classification rate of 65%. The best results were obtained using complex representation and Fourier/Zernike methods. In clinical practice, the preliminary diagnosis is based mainly on the visual inspection of the oral cavity, using both color, shape and texture as differentiating parameters. This study showed that machine analysis of shape could also play an important part in diagnosis and decisions regarding future treatment.

  15. An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing1[OPEN

    PubMed Central

    Lucas, Meriah K.

    2017-01-01

    Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes. PMID:28213559

  16. An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing.

    PubMed

    Hackett, Justin B; Shi, Xiaowen; Kobylarz, Amy T; Lucas, Meriah K; Wessendorf, Ryan L; Hines, Kevin M; Bentolila, Stephane; Hanson, Maureen R; Lu, Yan

    2017-04-01

    Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes.

  17. Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability

    PubMed Central

    Kang, Yilin; Baker, Michael James; Liem, Michael; Louber, Jade; McKenzie, Matthew; Atukorala, Ishara; Ang, Ching-Seng; Keerthikumar, Shivakumar; Mathivanan, Suresh; Stojanovski, Diana

    2016-01-01

    The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms. DOI: http://dx.doi.org/10.7554/eLife.17463.001 PMID:27554484

  18. Fuzzy nodes recognition based on spectral clustering in complex networks

    NASA Astrophysics Data System (ADS)

    Ma, Yang; Cheng, Guangquan; Liu, Zhong; Xie, Fuli

    2017-01-01

    In complex networks, information regarding the nodes is usually incomplete because of the effects of interference, noise, and other factors. This results in parts of the network being blurred and some information having an unknown source. In this paper, a spectral clustering algorithm is used to identify fuzzy nodes and solve network reconstruction problems. By changing the fuzzy degree of placeholders, we achieve various degrees of credibility and accuracy for the restored network. Our approach is verified by experiments using open source datasets and simulated data.

  19. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

    PubMed Central

    Costa, Sara R.; Marek, Magdalena; Axelsen, Kristian B.; Theorin, Lisa; Pomorski, Thomas G.; López-Marqués, Rosa L.

    2016-01-01

    P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase. PMID:27048590

  20. Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex.

    PubMed

    Cai, Yong; Jin, Jingji; Swanson, Selene K; Cole, Michael D; Choi, Seung Hyuk; Florens, Laurence; Washburn, Michael P; Conaway, Joan W; Conaway, Ronald C

    2010-02-12

    Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811-823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.

  1. Chromatin-modifying complex component Nurf55/p55 associates with histones H3 and H4 and polycomb repressive complex 2 subunit Su(z)12 through partially overlapping binding sites.

    PubMed

    Nowak, Agnieszka J; Alfieri, Claudio; Stirnimann, Christian U; Rybin, Vladimir; Baudin, Florence; Ly-Hartig, Nga; Lindner, Doris; Müller, Christoph W

    2011-07-01

    Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.

  2. Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

    PubMed

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H

    2015-02-20

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Purification, crystallization and preliminary X-ray diffraction analysis of the non-ATPase subunit Nas6 in complex with the ATPase subunit Rpt3 of the 26S proteasome from Saccharomyces cerevisiae

    SciTech Connect

    Nakamura, Yoshihiro; Umehara, Takashi; Tanaka, Akiko; Horikoshi, Masami; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2007-03-01

    The complex of the non-ATPase subunit Nas6 with the C-terminal domain of the ATPase subunit Rpt3 of the 26S proteasome from S. cerevisiae was co-expressed in E. coli and purified to homogeneity. The crystals obtained from the protein complex diffracted to a resolution of 2.2 Å. The non-ATPase subunit Nas6, which is the human orthologue of gankyrin, was co-expressed with the C-terminal domain of the ATPase subunit Rpt3 of the yeast 26S proteasome in Escherichia coli, purified to near-homogeneity and crystallized using the hanging-drop vapour-diffusion method. The protein crystallized in space group P2{sub 1}, with unit-cell parameters a = 60.38, b = 100.22, c = 72.20 Å, β = 94.70° and with three Nas6–Rpt3C molecules per asymmetric unit. The crystal diffracted to beyond 2.2 Å resolution using synchrotron radiation.

  4. The Effects of Semantic Transparency and Base Frequency on the Recognition of English Complex Words

    ERIC Educational Resources Information Center

    Xu, Joe; Taft, Marcus

    2015-01-01

    A visual lexical decision task was used to examine the interaction between base frequency (i.e., the cumulative frequencies of morphologically related forms) and semantic transparency for a list of derived words. Linear mixed effects models revealed that high base frequency facilitates the recognition of the complex word (i.e., a "base…

  5. The Role of Derivative Suffix Productivity in the Visual Word Recognition of Complex Words

    ERIC Educational Resources Information Center

    Lázaro, Miguel; Sainz, Javier; Illera, Víctor

    2015-01-01

    In this article we present two lexical decision experiments that examine the role of base frequency and of derivative suffix productivity in visual recognition of Spanish words. In the first experiment we find that complex words with productive derivative suffixes result in lower response times than those with unproductive derivative suffixes.…

  6. The Effects of Semantic Transparency and Base Frequency on the Recognition of English Complex Words

    ERIC Educational Resources Information Center

    Xu, Joe; Taft, Marcus

    2015-01-01

    A visual lexical decision task was used to examine the interaction between base frequency (i.e., the cumulative frequencies of morphologically related forms) and semantic transparency for a list of derived words. Linear mixed effects models revealed that high base frequency facilitates the recognition of the complex word (i.e., a "base…

  7. Acoustic interference and recognition space within a complex assemblage of dendrobatid frogs.

    PubMed

    Amézquita, Adolfo; Flechas, Sandra Victoria; Lima, Albertina Pimentel; Gasser, Herbert; Hödl, Walter

    2011-10-11

    In species-rich assemblages of acoustically communicating animals, heterospecific sounds may constrain not only the evolution of signal traits but also the much less-studied signal-processing mechanisms that define the recognition space of a signal. To test the hypothesis that the recognition space is optimally designed, i.e., that it is narrower toward the species that represent the higher potential for acoustic interference, we studied an acoustic assemblage of 10 diurnally active frog species. We characterized their calls, estimated pairwise correlations in calling activity, and, to model the recognition spaces of five species, conducted playback experiments with 577 synthetic signals on 531 males. Acoustic co-occurrence was not related to multivariate distance in call parameters, suggesting a minor role for spectral or temporal segregation among species uttering similar calls. In most cases, the recognition space overlapped but was greater than the signal space, indicating that signal-processing traits do not act as strictly matched filters against sounds other than homospecific calls. Indeed, the range of the recognition space was strongly predicted by the acoustic distance to neighboring species in the signal space. Thus, our data provide compelling evidence of a role of heterospecific calls in evolutionarily shaping the frogs' recognition space within a complex acoustic assemblage without obvious concomitant effects on the signal.

  8. Structural basis for the wobbler mouse neurodegenerative disorder caused by mutation in the Vps54 subunit of the GARP complex

    PubMed Central

    Pérez-Victoria, F. Javier; Abascal-Palacios, Guillermo; Tascón, Igor; Kajava, Andrey; Magadán, Javier G.; Pioro, Erik P.; Bonifacino, Juan S.; Hierro, Aitor

    2010-01-01

    The multisubunit Golgi-associated retrograde protein (GARP) complex is required for tethering and fusion of endosome-derived transport vesicles to the trans-Golgi network. Mutation of leucine-967 to glutamine in the Vps54 subunit of GARP is responsible for spinal muscular atrophy in the wobbler mouse, an animal model of amyotrophic lateral sclerosis. The crystal structure at 1.7 Å resolution of the mouse Vps54 C-terminal fragment harboring leucine-967, in conjunction with comparative sequence analysis, reveals that Vps54 has a continuous α-helical bundle organization similar to that of other multisubunit tethering complexes. The structure shows that leucine-967 is buried within the α-helical bundle through predominantly hydrophobic interactions that are critical for domain stability and folding in vitro. Mutation of this residue to glutamine does not prevent integration of Vps54 into the GARP complex but greatly reduces the half-life and levels of the protein in vivo. Severely reduced levels of mutant Vps54 and, consequently, of the whole GARP complex underlie the phenotype of the wobbler mouse. PMID:20615984

  9. Structural basis for the wobbler mouse neurodegenerative disorder caused by mutation in the Vps54 subunit of the GARP complex.

    PubMed

    Pérez-Victoria, F Javier; Abascal-Palacios, Guillermo; Tascón, Igor; Kajava, Andrey; Magadán, Javier G; Pioro, Erik P; Bonifacino, Juan S; Hierro, Aitor

    2010-07-20

    The multisubunit Golgi-associated retrograde protein (GARP) complex is required for tethering and fusion of endosome-derived transport vesicles to the trans-Golgi network. Mutation of leucine-967 to glutamine in the Vps54 subunit of GARP is responsible for spinal muscular atrophy in the wobbler mouse, an animal model of amyotrophic lateral sclerosis. The crystal structure at 1.7 A resolution of the mouse Vps54 C-terminal fragment harboring leucine-967, in conjunction with comparative sequence analysis, reveals that Vps54 has a continuous alpha-helical bundle organization similar to that of other multisubunit tethering complexes. The structure shows that leucine-967 is buried within the alpha-helical bundle through predominantly hydrophobic interactions that are critical for domain stability and folding in vitro. Mutation of this residue to glutamine does not prevent integration of Vps54 into the GARP complex but greatly reduces the half-life and levels of the protein in vivo. Severely reduced levels of mutant Vps54 and, consequently, of the whole GARP complex underlie the phenotype of the wobbler mouse.

  10. HIC1 interacts with a specific subunit of SWI/SNF complexes, ARID1A/BAF250A

    SciTech Connect

    Van Rechem, Capucine; Boulay, Gaylor; Leprince, Dominique

    2009-08-07

    HIC1, a tumor suppressor gene epigenetically silenced in many human cancers encodes a transcriptional repressor involved in regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. HIC1 is also implicated in growth control since it recruits BRG1, one of the two alternative ATPases (BRM or BRG1) of SWI/SNF chromatin-remodeling complexes to repress transcription of E2F1 in quiescent fibroblasts. Here, through yeast two-hybrid screening, we identify ARID1A/BAF250A, as a new HIC1 partner. ARID1A/BAF250A is one of the two mutually exclusive ARID1-containing subunits of SWI/SNF complexes which define subsets of complexes endowed with anti-proliferative properties. Co-immunoprecipitation assays in WI38 fibroblasts and in BRG1-/- SW13 cells showed that endogenous HIC1 and ARID1A proteins interact in a BRG1-dependent manner. Furthermore, we demonstrate that HIC1 does not interact with BRM. Finally, sequential chromatin immunoprecipitation (ChIP-reChIP) experiments demonstrated that HIC1 represses E2F1 through the recruitment of anti-proliferative SWI/SNF complexes containing ARID1A.

  11. Recognition of thymine in DNA bulges by a Zn(II) macrocyclic complex.

    PubMed

    del Mundo, Imee Marie A; Fountain, Matthew A; Morrow, Janet R

    2011-08-14

    A Zn(II) macrocyclic complex with appended quinoline is a bifunctional recognition agent that uses both the Zn(II) center and the pendent aromatic group to bind to thymine in bulges with good selectivity over DNA containing G, C or A bulges. Spectroscopic studies show that the stem containing the bulge stays largely intact in a DNA hairpin with the Zn(II) complex bound to the thymine bulge. This journal is © The Royal Society of Chemistry 2011

  12. Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes.

    PubMed

    Zhang, Jingqing; Landry, Markita P; Barone, Paul W; Kim, Jong-Ho; Lin, Shangchao; Ulissi, Zachary W; Lin, Dahua; Mu, Bin; Boghossian, Ardemis A; Hilmer, Andrew J; Rwei, Alina; Hinckley, Allison C; Kruss, Sebastian; Shandell, Mia A; Nair, Nitish; Blake, Steven; Şen, Fatih; Şen, Selda; Croy, Robert G; Li, Deyu; Yum, Kyungsuk; Ahn, Jin-Ho; Jin, Hong; Heller, Daniel A; Essigmann, John M; Blankschtein, Daniel; Strano, Michael S

    2013-12-01

    Understanding molecular recognition is of fundamental importance in applications such as therapeutics, chemical catalysis and sensor design. The most common recognition motifs involve biological macromolecules such as antibodies and aptamers. The key to biorecognition consists of a unique three-dimensional structure formed by a folded and constrained bioheteropolymer that creates a binding pocket, or an interface, able to recognize a specific molecule. Here, we show that synthetic heteropolymers, once constrained onto a single-walled carbon nanotube by chemical adsorption, also form a new corona phase that exhibits highly selective recognition for specific molecules. To prove the generality of this phenomenon, we report three examples of heteropolymer-nanotube recognition complexes for riboflavin, L-thyroxine and oestradiol. In each case, the recognition was predicted using a two-dimensional thermodynamic model of surface interactions in which the dissociation constants can be tuned by perturbing the chemical structure of the heteropolymer. Moreover, these complexes can be used as new types of spatiotemporal sensors based on modulation of the carbon nanotube photoemission in the near-infrared, as we show by tracking riboflavin diffusion in murine macrophages.

  13. Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Zhang, Jingqing; Landry, Markita P.; Barone, Paul W.; Kim, Jong-Ho; Lin, Shangchao; Ulissi, Zachary W.; Lin, Dahua; Mu, Bin; Boghossian, Ardemis A.; Hilmer, Andrew J.; Rwei, Alina; Hinckley, Allison C.; Kruss, Sebastian; Shandell, Mia A.; Nair, Nitish; Blake, Steven; Şen, Fatih; Şen, Selda; Croy, Robert G.; Li, Deyu; Yum, Kyungsuk; Ahn, Jin-Ho; Jin, Hong; Heller, Daniel A.; Essigmann, John M.; Blankschtein, Daniel; Strano, Michael S.

    2013-12-01

    Understanding molecular recognition is of fundamental importance in applications such as therapeutics, chemical catalysis and sensor design. The most common recognition motifs involve biological macromolecules such as antibodies and aptamers. The key to biorecognition consists of a unique three-dimensional structure formed by a folded and constrained bioheteropolymer that creates a binding pocket, or an interface, able to recognize a specific molecule. Here, we show that synthetic heteropolymers, once constrained onto a single-walled carbon nanotube by chemical adsorption, also form a new corona phase that exhibits highly selective recognition for specific molecules. To prove the generality of this phenomenon, we report three examples of heteropolymer-nanotube recognition complexes for riboflavin, L-thyroxine and oestradiol. In each case, the recognition was predicted using a two-dimensional thermodynamic model of surface interactions in which the dissociation constants can be tuned by perturbing the chemical structure of the heteropolymer. Moreover, these complexes can be used as new types of spatiotemporal sensors based on modulation of the carbon nanotube photoemission in the near-infrared, as we show by tracking riboflavin diffusion in murine macrophages.

  14. Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia.

    PubMed

    Skibinski, Gaia; Parkinson, Nicholas J; Brown, Jeremy M; Chakrabarti, Lisa; Lloyd, Sarah L; Hummerich, Holger; Nielsen, Jørgen E; Hodges, John R; Spillantini, Maria Grazia; Thusgaard, Tove; Brandner, Sebastian; Brun, Arne; Rossor, Martin N; Gade, Anders; Johannsen, Peter; Sørensen, Sven Asger; Gydesen, Susanne; Fisher, Elizabeth M C; Collinge, John

    2005-08-01

    We have previously reported a large Danish pedigree with autosomal dominant frontotemporal dementia (FTD) linked to chromosome 3 (FTD3). Here we identify a mutation in CHMP2B, encoding a component of the endosomal ESCRTIII complex, and show that it results in aberrant mRNA splicing in tissue samples from affected members of this family. We also describe an additional missense mutation in an unrelated individual with FTD. Aberration in the endosomal ESCRTIII complex may result in FTD and neurodegenerative disease.

  15. Eukaryotic DNA replication: Orchestrated action of multi-subunit protein complexes.

    PubMed

    Kang, Sukhyun; Kang, Mi-Sun; Ryu, Eunjin; Myung, Kyungjae

    2017-05-01

    Genome duplication is an essential process to preserve genetic information between generations. The eukaryotic cell cycle is composed of functionally distinct phases: G1, S, G2, and M. One of the key replicative proteins that participate at every stage of DNA replication is the Mcm2-7 complex, a replicative helicase. In the G1 phase, inactive Mcm2-7 complexes are loaded on the replication origins by replication-initiator proteins, ORC and Cdc6. Two kinases, S-CDK and DDK, convert the inactive origin-loaded Mcm2-7 complex to an active helicase, the CMG complex in the S phase. The activated CMG complex begins DNA unwinding and recruits enzymes essential for DNA synthesis to assemble a replisome at the replication fork. After completion of DNA synthesis, the inactive CMG complex on the replicated DNA is removed from chromatin to terminate DNA replication. In this review, we will discuss the structure, function, and regulation of the molecular machines involved in each step of DNA replication. Copyright © 2017. Published by Elsevier B.V.

  16. MITOCHONDRIAL DISEASES PART I: MOUSE MODELS OF OXPHOS DEFICIENCIES CAUSED BY DEFECTS ON RESPIRATORY COMPLEX SUBUNITS OR ASSEMBLY FACTORS

    PubMed Central

    Torraco, Alessandra; Peralta, Susana; Iommarini, Luisa; Diaz, Francisca

    2015-01-01

    Mitochondrial disorders are the most common inborn errors of metabolism affecting the oxidative phosphorylation system (OXPHOS). Because the poor knowledge of the pathogenic mechanisms, a cure for these disorders is still unavailable and all the treatments currently in use are supportive more than curative. Therefore, in the past decade a great variety of mouse models have been developed to assess the in vivo function of several mitochondrial proteins involved in human diseases. Due to the genetic and physiological similarity to humans, mice represent reliable models to study the pathogenic mechanisms of mitochondrial disorders and are precious to test new therapeutic approaches. Here we summarize the features of several mouse models of mitochondrial diseases directly related to defects in subunits of the OXPHOS complexes or in assembly factors. We discuss how these models recapitulate many human conditions and how they have contributed to the understanding of mitochondrial function in health and disease. PMID:25660179

  17. Asymmetric nature of two subunits of RAD18, a RING-type ubiquitin ligase E3, in the human RAD6A-RAD18 ternary complex.

    PubMed

    Masuda, Yuji; Suzuki, Miki; Kawai, Hidehiko; Suzuki, Fumio; Kamiya, Kenji

    2012-02-01

    RAD18, a RING-type ubiquitin ligase (E3) that plays an essential role in post-replication repair, possesses distinct domains named RING, UBZ, SAP and the RAD6-binding domain (R6BD) and forms a dimer. RAD6, an ubiquitin-conjugating enzyme (E2), stably associates with R6BD in the C-terminal portion. In this study, we established a method to distinguish between the two subunits of RAD18 by introduction of different tags, and analyzed mutant complexes. Our results, surprisingly, demonstrate that RAD6A and RAD18 form a ternary complex, RAD6A-(RAD18)(2) and the presence of only one R6BD in the two RAD18 subunits is sufficient for ternary complex formation and the ligase activity. Interestingly, ligase activity of a mutant dimer lacking both R6BDs is not restored even with large amounts of RAD6A added in solution, suggesting a requirement for precise juxtaposition via interaction with R6BD. We further show that mutations in both subunits of either RING or SAP, but not UBZ, strongly reduce ligase activity, although inactivation in only one of two subunits is without effect. These results suggest an asymmetric nature of the two RAD18 subunits in the complex.

  18. Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly.

    PubMed Central

    Lessard, I A; Perham, R N

    1995-01-01

    The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex. Images Figure 2 Figure 3 Figure 4 Figure 6 PMID:7702567

  19. The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis

    PubMed Central

    Haye, Joanna E.; Gammie, Alison E.

    2015-01-01

    During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/β to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles. PMID:26684201

  20. Basic and complex emotion recognition in children with autism: cross-cultural findings.

    PubMed

    Fridenson-Hayo, Shimrit; Berggren, Steve; Lassalle, Amandine; Tal, Shahar; Pigat, Delia; Bölte, Sven; Baron-Cohen, Simon; Golan, Ofer

    2016-01-01

    Children with autism spectrum conditions (ASC) have emotion recognition deficits when tested in different expression modalities (face, voice, body). However, these findings usually focus on basic emotions, using one or two expression modalities. In addition, cultural similarities and differences in emotion recognition patterns in children with ASC have not been explored before. The current study examined the similarities and differences in the recognition of basic and complex emotions by children with ASC and typically developing (TD) controls across three cultures: Israel, Britain, and Sweden. Fifty-five children with high-functioning ASC, aged 5-9, were compared to 58 TD children. On each site, groups were matched on age, sex, and IQ. Children were tested using four tasks, examining recognition of basic and complex emotions from voice recordings, videos of facial and bodily expressions, and emotional video scenarios including all modalities in context. Compared to their TD peers, children with ASC showed emotion recognition deficits in both basic and complex emotions on all three modalities and their integration in context. Complex emotions were harder to recognize, compared to basic emotions for the entire sample. Cross-cultural agreement was found for all major findings, with minor deviations on the face and body tasks. Our findings highlight the multimodal nature of ER deficits in ASC, which exist for basic as well as complex emotions and are relatively stable cross-culturally. Cross-cultural research has the potential to reveal both autism-specific universal deficits and the role that specific cultures play in the way empathy operates in different countries.

  1. Artificial Neural Network for the Prediction of Tyrosine-Based Sorting Signal Recognition by Adaptor Complexes

    PubMed Central

    Mukherjee, Debarati; Hanna, Claudia B.; Aguilar, R. Claudio

    2012-01-01

    Sorting of transmembrane proteins to various intracellular compartments depends on specific signals present within their cytosolic domains. Among these sorting signals, the tyrosine-based motif (YXXØ) is one of the best characterized and is recognized by μ-subunits of the four clathrin-associated adaptor complexes (AP-1 to AP-4). Despite their overlap in specificity, each μ-subunit has a distinct sequence preference dependent on the nature of the X-residues. Moreover, combinations of these residues exert cooperative or inhibitory effects towards interaction with the various APs. This complexity makes it impossible to predict a priori, the specificity of a given tyrosine-signal for a particular μ-subunit. Here, we describe the results obtained with a computational approach based on the Artificial Neural Network (ANN) paradigm that addresses the issue of tyrosine-signal specificity, enabling the prediction of YXXØ-μ interactions with accuracies over 90%. Therefore, this approach constitutes a powerful tool to help predict mechanisms of intracellular protein sorting. PMID:22505811

  2. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

    PubMed

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  3. Med1 Subunit of the Mediator Complex in Nuclear Receptor-Regulated Energy Metabolism, Liver Regeneration, and Hepatocarcinogenesis

    PubMed Central

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K.

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  4. Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice

    PubMed Central

    Jia, Yuzhi; Chang, Hsiang-Chun; Schipma, Matthew J.; Liu, Jing; Shete, Varsha; Liu, Ning; Sato, Tatsuya; Thorp, Edward B.; Barger, Philip M.; Zhu, Yi-Jun; Viswakarma, Navin; Kanwar, Yashpal S.; Ardehali, Hossein; Thimmapaya, Bayar; Reddy, Janardan K.

    2016-01-01

    Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart

  5. Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice.

    PubMed

    Jia, Yuzhi; Chang, Hsiang-Chun; Schipma, Matthew J; Liu, Jing; Shete, Varsha; Liu, Ning; Sato, Tatsuya; Thorp, Edward B; Barger, Philip M; Zhu, Yi-Jun; Viswakarma, Navin; Kanwar, Yashpal S; Ardehali, Hossein; Thimmapaya, Bayar; Reddy, Janardan K

    2016-01-01

    Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart

  6. Developmental Regulation of the Tetrahymena thermophila Origin Recognition Complex

    PubMed Central

    Lee, Po-Hsuen; Meng, Xiangzhou; Kapler, Geoffrey M.

    2015-01-01

    The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development. PMID:25569357

  7. Cell biology of the BLOC-1 complex subunit dysbindin, a schizophrenia susceptibility gene.

    PubMed

    Mullin, Ariana P; Gokhale, Avanti; Larimore, Jennifer; Faundez, Victor

    2011-08-01

    There is growing interest in the biology of dysbindin and its genetic locus (DTNBP1) due to genetic variants associated with an increased risk of schizophrenia. Reduced levels of dysbindin mRNA and protein in the hippocampal formation of schizophrenia patients further support involvement of this locus in disease risk. Here, we discuss phylogenetically conserved dysbindin molecular interactions that define its contribution to the assembly of the biogenesis of lysosome-related organelles complex-1 (BLOC-1). We explore fundamental cellular processes where dysbindin and the dysbindin-containing BLOC-1 complex are implicated. We propose that cellular, tissue, and system neurological phenotypes from dysbindin deficiencies in model genetic organisms, and likely individuals affected with schizophrenia, emerge from abnormalities in few core cellular mechanisms controlled by BLOC-1-dysbindin-containing complex rather than from defects in dysbindin itself.

  8. The Evolution of the Four Subunits of Voltage-Gated Calcium Channels: Ancient Roots, Increasing Complexity, and Multiple Losses

    PubMed Central

    Moran, Yehu; Zakon, Harold H.

    2014-01-01

    The alpha subunits of voltage-gated calcium channels (Cavs) are large transmembrane proteins responsible for crucial physiological processes in excitable cells. They are assisted by three auxiliary subunits that can modulate their electrical behavior. Little is known about the evolution and roles of the various subunits of Cavs in nonbilaterian animals and in nonanimal lineages. For this reason, we mapped the phyletic distribution of the four channel subunits and reconstructed their phylogeny. Although alpha subunits have deep evolutionary roots as ancient as the split between plants and opistokonths, beta subunits appeared in the last common ancestor of animals and their close-relatives choanoflagellates, gamma subunits are a bilaterian novelty and alpha2/delta subunits appeared in the lineage of Placozoa, Cnidaria, and Bilateria. We note that gene losses were extremely common in the evolution of Cavs, with noticeable losses in multiple clades of subfamilies and also of whole Cav families. As in vertebrates, but not protostomes, Cav channel genes duplicated in Cnidaria. We characterized by in situ hybridization the tissue distribution of alpha subunits in the sea anemone Nematostella vectensis, a nonbilaterian animal possessing all three Cav subfamilies common to Bilateria. We find that some of the alpha subunit subtypes exhibit distinct spatiotemporal expression patterns. Further, all six sea anemone alpha subunit subtypes are conserved in stony corals, which separated from anemones 500 MA. This unexpected conservation together with the expression patterns strongly supports the notion that these subtypes carry unique functional roles. PMID:25146647

  9. Binding energies of nucleobase complexes: Relevance to homology recognition of DNA

    NASA Astrophysics Data System (ADS)

    León, Sergio Cruz; Prentiss, Mara; Fyta, Maria

    2016-06-01

    The binding energies of complexes of DNA nucleobase pairs are evaluated using quantum mechanical calculations at the level of dispersion corrected density functional theory. We begin with Watson-Crick base pairs of singlets, duplets, and triplets and calculate their binding energies. At a second step, mismatches are incorporated into the Watson-Crick complexes in order to evaluate the variation in the binding energy with respect to the canonical Watson-Crick pairs. A linear variation of this binding energy with the degree of mismatching is observed. The binding energies for the duplets and triplets containing mismatches are further compared to the energies of the respective singlets in order to assess the degree of collectivity in these complexes. This study also suggests that mismatches do not considerably affect the energetics of canonical base pairs. Our work is highly relevant to the recognition process in DNA promoted through the RecA protein and suggests a clear distinction between recognition in singlets, and recognition in duplets or triplets. Our work assesses the importance of collectivity in the homology recognition of DNA.

  10. Direct Localization of the 51 kDa and 24 kDa Subunits of Mitochondrial Complex I by Three-dimensional Difference Imaging

    PubMed Central

    Clason, Todd; Zickermann, Volker; Ruiz, Teresa; Brandt, Ulrich; Radermacher, Michael

    2009-01-01

    Complex I is the largest complex in the respiratory chain, and the least understood. We have determined the three-dimensional structure of complex I from Yarrowia lipolytica lacking the flavoprotein part of the N-module, which consists of the 51 kDa (NUBM) and the 24 kDa (NUHM) subunits. The reconstruction was determined by three-dimensional electron microscopy of single particles. A comparison to our earlier reconstruction of the complete Y. lipolytica complex I clearly assigns the two flavoprotein subunits to an outer lobe of the peripheral arm of complex I. Localizing the two subunits allowed us to fit the X-ray structure of the hydrophilic fragment of complex I from Thermus thermophilus. The fit that is most consistent with previous immuno-electron microscopic data predicts that the ubiquinone reducing catalytic center resides in the second peripheral lobe, while the 75 kDa subunit is placed near the previously seen connection between the peripheral arm and the membrane arm protrusions. PMID:17591445

  11. Crystal structure of subunit VPS25 of the endosomal trafficking complex ESCRT-II

    PubMed Central

    Wernimont, Amy K; Weissenhorn, Winfried

    2004-01-01

    Background Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB) destined for lysosomal degradation. Successive assemblies of Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II and III) largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity. Results Here we report the crystal structure of Vps25 at 3.1 Å resolution. Vps25 crystallizes in a dimeric form and each monomer is composed of two winged helix domains arranged in tandem. Structural comparisons detect no conformational changes between unliganded Vps25 and Vps25 within the ESCRT-II complex composed of two Vps25 copies and one copy each of Vps22 and Vps36 [1,2]. Conclusions Our structural analyses present a framework for studying Vps25 interactions with ESCRT-I and ESCRT-III partners. Winged helix domain containing proteins have been implicated in nucleic acid binding and it remains to be determined whether Vps25 has a similar activity which might play a role in the proposed transcriptional control exerted by Vps25 and/or the whole ESCRT-II complex. PMID:15579210

  12. Energy based approach for understanding the recognition mechanism in protein-protein complexes.

    PubMed

    Gromiha, M Michael; Yokota, Kiyonobu; Fukui, Kazuhiko

    2009-12-01

    Protein-protein interactions play an essential role in the regulation of various cellular processes. Understanding the recognition mechanism of protein-protein complexes is a challenging task in molecular and computational biology. In this work, we have developed an energy based approach for identifying the binding sites and important residues for binding in protein-protein complexes. The new approach is different from the traditional distance based contacts in which the repulsive interactions are treated as binding sites as well as the contacts within a specific cutoff have been treated in the same way. We found that the residues and residue-pairs with charged and aromatic side chains are important for binding. These residues influence to form cation-, electrostatic and aromatic interactions. Our observation has been verified with the experimental binding specificity of protein-protein complexes and found good agreement with experiments. Based on these results we have proposed a novel mechanism for the recognition of protein-protein complexes: the charged and aromatic residues in receptor and ligand initiate recognition by making suitable interactions between them; the neighboring hydrophobic residues assist the stability of complex along with other hydrogen bonding partners by the polar residues. Further, the propensity of residues in the binding sites of receptors and ligands, atomic contributions and the influence on secondary structure will be discussed.

  13. Crystal structure of AcrB in complex with a single transmembrane subunit reveals another twist.

    PubMed

    Törnroth-Horsefield, Susanna; Gourdon, Pontus; Horsefield, Rob; Brive, Lars; Yamamoto, Natsuko; Mori, Hirotada; Snijder, Arjan; Neutze, Richard

    2007-12-01

    Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.

  14. A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

    PubMed

    Herbert, Kristina M; Sarkar, Susanta K; Mills, Maria; Delgado De la Herran, Hilda C; Neuman, Keir C; Steitz, Joan A

    2016-02-01

    During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.

  15. Isolation and characterization of human orthologs of yeast CCR4–NOT complex subunits

    PubMed Central

    Albert, Thomas K.; Lemaire, Marc; Berkum, Nynke L. van; Gentz, Reiner; Collart, Martine A.; Timmers, H. Th. Marc

    2000-01-01

    The yeast CCR4–NOT protein complex is a global regulator of RNA polymerase II transcription. It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional proteins like yeast CAF1. Here we report the isolation of cDNAs encoding human NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF. Analysis of their mRNA levels in different human tissues reveals a common ubiquitous expression pattern. A multitude of two-hybrid interactions among the human cDNAs suggest that their encoded proteins also form a complex in mammalian cells. Functional conservation of these proteins throughout evolution is supported by the observation that the isolated human NOT3 and NOT4 cDNAs can partially complement corresponding not mutations in yeast. Interestingly, human CALIF is highly homologous to, although clearly different from, a recently described human CAF1 protein. Conserved interactions of this factor with both NOT and CCR4 proteins and co-immunoprecipitation experiments suggest that CALIF is a bona fide component of the human CCR4–NOT complex. PMID:10637334

  16. The Arabidopsis Mediator Complex Subunit16 Is a Key Component of Basal Resistance against the Necrotrophic Fungal Pathogen Sclerotinia sclerotiorum.

    PubMed

    Wang, Chenggang; Yao, Jin; Du, Xuezhu; Zhang, Yanping; Sun, Yijun; Rollins, Jeffrey A; Mou, Zhonglin

    2015-09-01

    Although Sclerotinia sclerotiorum is a devastating necrotrophic fungal plant pathogen in agriculture, the virulence mechanisms utilized by S. sclerotiorum and the host defense mechanisms against this pathogen have not been fully understood. Here, we report that the Arabidopsis (Arabidopsis thaliana) Mediator complex subunit MED16 is a key component of basal resistance against S. sclerotiorum. Mutants of MED16 are markedly more susceptible to S. sclerotiorum than mutants of 13 other Mediator subunits, and med16 has a much stronger effect on S. sclerotiorum-induced transcriptome changes compared with med8, a mutation not altering susceptibility to S. sclerotiorum. Interestingly, med16 is also more susceptible to S. sclerotiorum than coronatine-insensitive1-1 (coi1-1), which is the most susceptible mutant reported so far. Although the jasmonic acid (JA)/ethylene (ET) defense pathway marker gene PLANT DEFENSIN1.2 (PDF1.2) cannot be induced in either med16 or coi1-1, basal transcript levels of PDF1.2 in med16 are significantly lower than in coi1-1. Furthermore, ET-induced suppression of JA-activated wound responses is compromised in med16, suggesting a role for MED16 in JA-ET cross talk. Additionally, MED16 is required for the recruitment of RNA polymerase II to PDF1.2 and OCTADECANOID-RESPONSIVE ARABIDOPSIS ETHYLENE/ETHYLENE-RESPONSIVE FACTOR59 (ORA59), two target genes of both JA/ET-mediated and the transcription factor WRKY33-activated defense pathways. Finally, MED16 is physically associated with WRKY33 in yeast and in planta, and WRKY33-activated transcription of PDF1.2 and ORA59 as well as resistance to S. sclerotiorum depends on MED16. Taken together, these results indicate that MED16 regulates resistance to S. sclerotiorum by governing both JA/ET-mediated and WRKY33-activated defense signaling in Arabidopsis.

  17. Metal-organic complex-functionalized protein nanopore sensor for aromatic amino acids chiral recognition.

    PubMed

    Guo, Yanli; Niu, Aihua; Jian, Feifei; Wang, Ying; Yao, Fujun; Wei, Yongfeng; Tian, Lei; Kang, Xiaofeng

    2017-03-27

    Chiral recognition at single-molecule level for small active molecules is important, as exhibited by many nanostructures and molecular assemblies in biological systems, but it presents a significant challenge. We report a simple and rapid sensing strategy to discriminate all enantiomers of natural aromatic amino acids (AAA) using a metal-organic complex-functionalized protein nanopore, in which a chiral recognition element and a chiral recognition valve were equipped. A trifunctional molecule, heptakis-(6-deoxy-6-amino)-β-cyclodextrin (am7βCD), was non-covalently lodged within the nanopore of an α-hemolysin (αHL) mutant, (M113R)7-αHL. Copper(ii) ion reversibly bonds to the amino group of am7βCD to form an am7βCD-Cu(II) complex, which allowed chiral recognition for each enantiomer in the mixture of AAA by distinct current signals. The Cu(II) plugging valve plays a crucial rule that holds chiral molecules in the nanocavity for a sufficient registering time. Importantly, six enantiomers of all nature AAA could be simultaneously recognized at one time. Enantiomeric excess (ee) could also be accurately detected by this approach. It should be possible to generalize this approach for sensing of other chiral molecules.

  18. Pyruvate dehydrogenase complex: mRNA and protein expression patterns of E1α subunit genes in human spermatogenesis.

    PubMed

    Pinheiro, Ana; Silva, Maria João; Graça, Inês; Silva, Joaquina; Sá, Rosália; Sousa, Mário; Barros, Alberto; Tavares de Almeida, Isabel; Rivera, Isabel

    2012-09-10

    During spermatogenesis, germ cells undergo a complex process of cell differentiation and morphological restructuring, which depends on the coordinated expression of different genes. Some vital examples are those involved in cell energy metabolism, namely the genes encoding the E1α subunit of pyruvate dehydrogenase complex: the somatic PDHA1 (X-linked) and the testis-specific PDHA2 (autosomal). There are no data related to the study at the RNA and protein levels of PDHA genes during human spermatogenesis. The present study aimed to describe the mRNA and protein expression patterns of the human PDHA genes during spermatogenesis. Expression profiles of the PDHA1 and PDHA2 genes were characterized using different human tissues and cells. Diploid and haploid germ cells fractions were obtained from testis tissues. The mRNA profiles were analyzed by quantitative RT-PCR, whereas the protein profiles were evaluated by immunohistochemistry, western blotting and two-dimensional electrophoresis. Expression of the PDHA1 gene was found in all somatic cells, whereas expression of PDHA2 gene was restricted to germ cells. The switch from X-linked to autosomic gene expression occurred in spermatocytes. Data suggest the activation of PDHA2 gene expression is most probably a mechanism to ensure the continued expression of the protein, thus allowing germ cell viability and functionality.

  19. Probability Fluxes and Transition Paths in a Markovian Model Describing Complex Subunit Cooperativity in HCN2 Channels

    PubMed Central

    Benndorf, Klaus; Kusch, Jana; Schulz, Eckhard

    2012-01-01

    Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are voltage-gated tetrameric cation channels that generate electrical rhythmicity in neurons and cardiomyocytes. Activation can be enhanced by the binding of adenosine-3′,5′-cyclic monophosphate (cAMP) to an intracellular cyclic nucleotide binding domain. Based on previously determined rate constants for a complex Markovian model describing the gating of homotetrameric HCN2 channels, we analyzed probability fluxes within this model, including unidirectional probability fluxes and the probability flux along transition paths. The time-dependent probability fluxes quantify the contributions of all 13 transitions of the model to channel activation. The binding of the first, third and fourth ligand evoked robust channel opening whereas the binding of the second ligand obstructed channel opening similar to the empty channel. Analysis of the net probability fluxes in terms of the transition path theory revealed pronounced hysteresis for channel activation and deactivation. These results provide quantitative insight into the complex interaction of the four structurally equal subunits, leading to non-equality in their function. PMID:23093920

  20. Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

    PubMed Central

    Leigh, Kendra E.; Sharma, Mayuri; Mansueto, My Sam; Boeszoermenyi, Andras; Filman, David J.; Hogle, James M.; Wagner, Gerhard; Coen, Donald M.; Arthanari, Haribabu

    2015-01-01

    Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1–168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50–UL53 binding in vitro, eliminated UL50–UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein–protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target. PMID:26150520

  1. Mutations in Two Genes Encoding Different Subunits of a Receptor Signaling Complex Result in an Identical Disease Phenotype

    PubMed Central

    Paloneva, Juha; Manninen, Tuula; Christman, Grant; Hovanes, Karine; Mandelin, Jami; Adolfsson, Rolf; Bianchin, Marino; Bird, Thomas; Miranda, Roxana; Salmaggi, Andrea; Tranebjærg, Lisbeth; Konttinen, Yrjö; Peltonen, Leena

    2002-01-01

    Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as “Nasu-Hakola disease,” is a globally distributed recessively inherited disease leading to death during the 5th decade of life and is characterized by early-onset progressive dementia and bone cysts. Elsewhere, we have identified PLOSL mutations in TYROBP (DAP12), which codes for a membrane receptor component in natural-killer and myeloid cells, and also have identified genetic heterogeneity in PLOSL, with some patients carrying no mutations in TYROBP. Here we complete the molecular pathology of PLOSL by identifying TREM2 as the second PLOSL gene. TREM2 forms a receptor signaling complex with TYROBP and triggers activation of the immune responses in macrophages and dendritic cells. Patients with PLOSL have no defects in cell-mediated immunity, suggesting a remarkable capacity of the human immune system to compensate for the inactive TYROBP-mediated activation pathway. Our data imply that the TYROBP-mediated signaling pathway plays a significant role in human brain and bone tissue and provide an interesting example of how mutations in two different subunits of a multisubunit receptor complex result in an identical human disease phenotype. PMID:12080485

  2. Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication.

    PubMed

    Leigh, Kendra E; Sharma, Mayuri; Mansueto, My Sam; Boeszoermenyi, Andras; Filman, David J; Hogle, James M; Wagner, Gerhard; Coen, Donald M; Arthanari, Haribabu

    2015-07-21

    Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.

  3. p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

    PubMed Central

    Tinsley, J H; Minke, P F; Bruno, K S; Plamann, M

    1996-01-01

    Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae. Images PMID:8744947

  4. Multistep Processing of an Insertion Sequence in an Essential Subunit of the Chloroplast ClpP Complex*

    PubMed Central

    Derrien, Benoit; Majeran, Wojciech; Wollman, Francis-André; Vallon, Olivier

    2009-01-01

    In Chlamydomonas reinhardtii, the clpP1 chloroplast gene encoding one of the catalytic subunits of the ClpP protease complex contains a large in-frame insertion sequence (IS1). Based on the Escherichia coli ClpP structure, IS1 is predicted to protrude at the apical surface of the complex, likely influencing the interaction of the catalytic core with ClpC/HSP100 chaperones. Immunoblotting with an anti-ClpP1 antibody detected two immunoreactive forms of ClpP1: ClpP1H (59 kDa) and ClpP1L (25 kDa). It has been proposed that IS1 is a new type of protein intron (different from inteins). By studying transformants harboring mutations at the predicted borders of IS1 and tags at the C terminus of ClpP1 (tandem affinity purification tag, His tag, Strep·Tag) or within the IS1 sequence (3-hemagglutinin tag), we show that IS1 is not a protein intron and that ClpP1L results from endoproteolytic cleavage inside IS1. Processing sites have been identified in the middle of IS1 and near its C terminus. The sites can be mutated without abolishing processing. PMID:19346247

  5. Insights into the Replisome from the Structure of a Ternary Complex of the DNA Polymerase III [alpha]-Subunit

    SciTech Connect

    Wing, R.A.; Bailey, S.; Steitz, T.A.

    2009-03-27

    The crystal structure of the catalytic {alpha}-subunit of the DNA polymerase III (PolIII{alpha}) holoenzyme bound to primer-template DNA and an incoming deoxy-nucleoside 5{prime}-triphosphate has been determined at 4.6-{angstrom} resolution. The polymerase interacts with the sugar-phosphate backbone of the DNA across its minor groove, which is made possible by significant movements of the thumb, finger, and {beta}-binding domains relative to their orientations in the unliganded polymerase structure. Additionally, the DNA and incoming nucleotide are bound to the active site of PolIII{alpha} nearly identically as they are in their complex with DNA polymerase {beta}, thereby proving that the eubacterial replicating polymerase, but not the eukaryotic replicating polymerase, is homologous to DNA polymerase {beta}. Finally, superimposing a recent structure of the clamp bound to DNA on this PolIII{alpha} complex with DNA places a loop of the {beta}-binding domain into the appropriate clamp cleft and supports a mechanism of polymerase switching.

  6. The putative SWI/SNF complex subunit BRAHMA activates flower homeotic genes in Arabidopsis thaliana.

    PubMed

    Hurtado, Lidia; Farrona, Sara; Reyes, Jose C

    2006-09-01

    Arabidopsis thaliana BRAHMA (BRM, also called AtBRM) is a SNF2 family protein homolog of Brahma, the ATPase of the Drosophila SWI/SNF complex involved in chromatin remodeling during transcription. Here we show that, in contrast to its Drosophila counterpart, BRM is not an essential gene. Thus, homozygous BRM loss of function mutants are viable but exhibit numerous defects including dwarfism, altered leaf and root development and several reproduction defects. The analysis of the progeny of self-fertilized heterozygous brm plants and reciprocal crosses between heterozygous and wild type plants indicated that disruption of BRM reduced both male and female gametophyte transmission. This was consistent with the presence of aborted ovules in the self-fertilized heterozygous flowers that contained arrested embryos predominantly at the two terminal cells stage. Furthermore, brm homozygous mutants were completely sterile. Flowers of brm loss-of-function mutants have several developmental abnormalities, including homeotic transformations in the second and third floral whorls. In accordance with these results, brm mutants present reduced levels of APETALA2, APETALA3, PISTILLATA and NAC-LIKE, ACTIVATED BY AP3/PI. We have previously shown that BRM strongly interacts with AtSWI3C. Now we extend our interaction studies demonstrating that BRM interacts weakly with AtSWI3B but not with AtSWI3A or AtSWI3D. In agreement with these results, the phenotype described in this study for brm plants is very similar to that previously described for the AtSWI3C mutant plants, suggesting that both proteins participate in the same genetic pathway or form a molecular complex.

  7. Synthesis and characterization of tris(heteroleptic) Ru(II) complexes bearing styryl subunits.

    PubMed

    Myahkostupov, Mykhaylo; Castellano, Felix N

    2011-10-03

    We have developed and optimized a well-controlled and refined methodology for the synthesis of substituted π-conjugated 4,4'-styryl-2,2'-bipyridine ligands and also adapted the tris(heteroleptic) synthetic approach developed by Mann and co-workers to produce two new representative Ru(II)-based complexes bearing the metal oxide surface-anchoring precursor 4,4'-bis[E-(p-methylcarboxy-styryl)]-2,2'-bipyridine. The two targeted Ru(II) complexes, (4,4'-dimethyl-2,2'-bipyridine)(4,4'-di-tert-butyl-2,2'-bipyridine)(4,4'-bis[E-(p-methylcarboxy-styryl)]-2,2'-bipyridine) ruthenium(II) hexafluorophosphate, [Ru(dmbpy)(dtbbpy)(p-COOMe-styryl-bpy)](PF(6))(2) (1) and (4,4'-dimethyl-2,2'-bipyridine)(4,4'-dinonyl-2,2'-bipyridine)(4,4'-bis[E-(p-methylcarboxy-styryl)]-2,2'-bipyridine) ruthenium(II) hexafluorophosphate, [Ru(dmbpy)(dnbpy)(p-COOMe-styryl-bpy)](PF(6))(2) (2) were obtained as analytically pure compounds in high overall yields (>50% after 5 steps) and were isolated without significant purification effort. In these tris(heteroleptic) molecules, NMR-based structural characterization became nontrivial as the coordinated ligand sets each sense profoundly distinct magnetic environments greatly complicating traditional 1D spectra. However, rational two-dimensional approaches based on both homo- and heteronuclear couplings were readily applied to these structures producing quite definitive analytical characterization and the associated methodology is described in detail. Preliminary photoluminescence and photochemical characterization of 1 and 2 strongly suggests that both molecules are energetically and kinetically suitable to serve as sensitizers in energy-relevant applications.

  8. Enhanced retinal modeling for face recognition and facial feature point detection under complex illumination conditions

    NASA Astrophysics Data System (ADS)

    Cheng, Yong; Li, Zuoyong; Jiao, Liangbao; Lu, Hong; Cao, Xuehong

    2016-07-01

    We improved classic retinal modeling to alleviate the adverse effect of complex illumination on face recognition and extracted robust image features. Our improvements on classic retinal modeling included three aspects. First, a combined filtering scheme was applied to simulate functions of horizontal and amacrine cells for accurate local illumination estimation. Second, we developed an optimal threshold method for illumination classification. Finally, we proposed an adaptive factor acquisition model based on the arctangent function. Experimental results on the combined Yale B; the Carnegie Mellon University poses, illumination, and expression; and the Labeled Face Parts in the Wild databases show that the proposed method can effectively alleviate illumination difference of images under complex illumination conditions, which is helpful for improving the accuracy of face recognition and that of facial feature point detection.

  9. VMA13 encodes a 54-kDa vacuolar H(+)-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae.

    PubMed

    Ho, M N; Hirata, R; Umemoto, N; Ohya, Y; Takatsuki, A; Stevens, T H; Anraku, Y

    1993-08-25

    Previous purifications and characterizations of the Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) have indicated that this enzyme is a multisubunit complex composed of at least eight subunits of 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We report the cloning and characterization of an additional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 gene. VMA13 was isolated by complementation of the growth phenotypes associated with the vma13 mutation, which was originally described as cls11 (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The nucleotide sequence of the VMA13 gene predicted a hydrophilic polypeptide with a calculated molecular mass of 54,415 daltons. The VMA13 54-kDa gene product resides on the vacuolar membrane and co-purified with the active V-ATPase complex. Characterization of a null vma13 mutant (delta vma13) revealed that the Vma13 polypeptide is essential for V-ATPase activity. However, the Vma13 polypeptide is not required for targeting of the other V-ATPase subunits (100-, 69-, 60-, 42-, 27-, or 17-kDa subunits) to the vacuolar membrane as shown by the association of these subunits with vacuolar membranes isolated from delta vma13 cells. The nature of the V-ATPase "complex" in delta vma13 mutant is, nevertheless, fundamentally different from the wild-type enzyme. This is evidenced by the fact that the inactive V-ATPase complex from delta vma13 cells is less stable than the wild-type enzyme. Taken together, these results indicate that VMA13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme complex.

  10. CIF-1, a shared subunit of the COP9/signalosome and eukaryotic initiation factor 3 complexes, regulates MEL-26 levels in the Caenorhabditis elegans embryo.

    PubMed

    Luke-Glaser, Sarah; Roy, Marcia; Larsen, Brett; Le Bihan, Thierry; Metalnikov, Pavel; Tyers, Mike; Peter, Matthias; Pintard, Lionel

    2007-06-01

    The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation.

  11. CIF-1, a Shared Subunit of the COP9/Signalosome and Eukaryotic Initiation Factor 3 Complexes, Regulates MEL-26 Levels in the Caenorhabditis elegans Embryo▿

    PubMed Central

    Luke-Glaser, Sarah; Roy, Marcia; Larsen, Brett; Le Bihan, Thierry; Metalnikov, Pavel; Tyers, Mike; Peter, Matthias; Pintard, Lionel

    2007-01-01

    The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation. PMID:17403899

  12. Interactions of age, ear, and stimulus complexity on dichotic digit recognition.

    PubMed

    Wilson, R H; Jaffe, M S

    1996-10-01

    The effect that the aging process has on recognition performance was studied using a hierarchy of 1-pair, 2-pair, 3-pair, and 4-pair dichotic digits (1, 2, 3, 4, 5, 6, 8, 9, and 10, male speaker). Two groups of right-handed subjects were studied: 20 adults < 30 years of age with normal hearing and 20 adults 60-75 years with mild-to-moderate hearing loss. Each of the multipaired sets contained the 72 possible 1-pair digit combinations in each presentation position with no digits repeated in a set. A three-way analysis of variance with repeated measures indicated three significant differences. First, as the complexity of the listening task increased from 1 pair to 2 pairs to 3 pairs to 4 pairs, recognition performance decreased systematically and significantly. The decreases in performance between the 1- and 4-paired conditions were larger for the left ear than for the right ear and were larger for the 60-75 years group than for the < 30 years group. Second, performance on the materials presented to the right ear was significantly better than performance on the materials presented to the left ear. Third, recognition performance by the < 30 years group was significantly better than recognition performance by the 60-75 years group. Recognition performance on the 1-pair condition was near maximum for both subject groups. Between the 1- and 4-paired conditions, recognition performance for materials presented to the left and right ears decreased 15.7 percent and 10.0 percent, respectively, in the < 30 years group and decreased 29.3 percent and 15.7 percent, respectively, in the 60-75 years group.

  13. Research of location method for billet recognition in complex production line scene

    NASA Astrophysics Data System (ADS)

    Hong, Hanyu; Yu, Zhejun; Zhang, Xiuhua

    2011-11-01

    Steel code location is the key point to realize billet detection and recognition in production line scene with complex illumination. However, due to high temperature and complex scene in the rolling line, the steel code location at the end of billet is quite different from optical character location with simple background and vehicle license plate location. In the process of billet detection and recognition, how to determine steel code target location at the end of billet from the complex illumination scene is first necessary in steel intelligent recognition system. In order to solve this problem, a novel method for steel code location is proposed in this paper. First of all, production line scene image is restrained by Mean Shift filtering and iterative segmentation filter, and then candidate character region can be found by clustering character connected domain with same features. At last, the quantitative model is established for candidate region and the statistical decision algorithm can be used to complete screening object region. The experimental results show that the proposed location method is very precise in most different scenes.

  14. The Med1 Subunit of the Mediator Complex Induces Liver Cell Proliferation and Is Phosphorylated by AMP Kinase*

    PubMed Central

    Viswakarma, Navin; Jia, Yuzhi; Bai, Liang; Gao, Qian; Lin, Bingliang; Zhang, Xiaohong; Misra, Parimal; Rana, Ajay; Jain, Sanjay; Gonzalez, Frank J.; Zhu, Yi-Jun; Thimmapaya, Bayar; Reddy, Janardan K.

    2013-01-01

    Mediator, a large multisubunit protein complex, plays a pivotal role in gene transcription by linking gene-specific transcription factors with the preinitiation complex and RNA polymerase II. In the liver, the key subunit of the Mediator complex, Med1, interacts with several nuclear receptors and transcription factors to direct gene-specific transcription. Conditional knock-out of Med1 in the liver showed that hepatocytes lacking Med1 did not regenerate following either partial hepatectomy or treatment with certain nuclear receptor activators and failed to give rise to tumors when challenged with carcinogens. We now report that the adenovirally driven overexpression of Med1 in mouse liver stimulates hepatocyte DNA synthesis with enhanced expression of DNA replication, cell cycle control, and liver-specific genes, indicating that Med1 alone is necessary and sufficient for liver cell proliferation. Importantly, we demonstrate that AMP-activated protein kinase (AMPK), an important cellular energy sensor, interacts with, and directly phosphorylates, Med1 in vitro at serine 656, serine 756, and serine 796. AMPK also phosphorylates Med1 in vivo in mouse liver and in cultured primary hepatocytes and HEK293 and HeLa cells. In addition, we demonstrate that PPARα activators increase AMPK-mediated Med1 phosphorylation in vivo. Inhibition of AMPK by compound C decreased hepatocyte proliferation induced by Med1 and also by the PPARα activators fenofibrate and Wy-14,643. Co-treatment with compound C attenuated PPARα activator-inducible fatty acid β-oxidation in liver. Our results suggest that Med1 phosphorylation by its association with AMPK regulates liver cell proliferation and fatty acid oxidation, most likely as a downstream effector of PPARα and AMPK. PMID:23943624

  15. Integrating computational methods and experimental data for understanding the recognition mechanism and binding affinity of protein-protein complexes.

    PubMed

    Gromiha, M Michael; Yugandhar, K

    2017-09-01

    Protein-protein interactions perform several functions inside the cell. Understanding the recognition mechanism and binding affinity of protein-protein complexes is a challenging problem in experimental and computational biology. In this review, we focus on two aspects (i) understanding the recognition mechanism and (ii) predicting the binding affinity. The first part deals with computational techniques for identifying the binding site residues and the contribution of important interactions for understanding the recognition mechanism of protein-protein complexes in comparison with experimental observations. The second part is devoted to the methods developed for discriminating high and low affinity complexes, and predicting the binding affinity of protein-protein complexes using three-dimensional structural information and just from the amino acid sequence. The overall view enhances our understanding of the integration of experimental data and computational methods, recognition mechanism of protein-protein complexes and the binding affinity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Individual odor recognition in procellariiform chicks: potential role for the major histocompatibility complex.

    PubMed

    O'Dwyer, Terence W; Nevitt, Gabrielle A

    2009-07-01

    Since the groundbreaking work of Wenzel, Bang, and Grubb in the 1960s, enormous progress has been made toward elucidating the sense of smell in procellariiform seabirds. Although it is now well established that adult procellariiforms use olfaction in many behaviors, such as for foraging, nest relocation, and mate recognition, the olfactory abilities of petrel chicks are less well understood. Recent studies have shown that petrel chicks can recognize prey-related odors and odors associated with their nest before leaving their burrow for the first time. The recognition of burrow odors by petrel chicks is unlikely to be used for homing, and we have suggested that chicks may be learning personal odors associated with the nest's occupants for use later in life in the context of kin recognition or mate choice. The source of personal odors in petrels is unknown. However, in other vertebrates, the major histocompatibility complex influences body odors, which in turn influence mating preferences. It is not currently known whether this highly polymorphic gene region influences body odors and individual recognition in the procellariiforms, but this could be a fruitful area of future research.

  17. Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa

    PubMed Central

    Rollins, MaryClare F.; Schuman, Jason T.; Paulus, Kirra; Bukhari, Habib S.T.; Wiedenheft, Blake

    2015-01-01

    The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1–4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine–cytosine base pairs (G–C/G–C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be double-stranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G–C/G–C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G–C/G–C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G–C/G–C base pairs and then samples adjacent target sequences for complementarity to the crRNA guide. PMID:25662606

  18. Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity.

    PubMed

    Garofano, Aurelio; Zwicker, Klaus; Kerscher, Stefan; Okun, Pamela; Brandt, Ulrich

    2003-10-24

    Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.

  19. Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.

    PubMed

    Ma, Xin; Lai, Lien B; Lai, Stella M; Tanimoto, Akiko; Foster, Mark P; Wysocki, Vicki H; Gopalan, Venkat

    2014-10-20

    We demonstrate that surface-induced dissociation (SID) coupled with ion mobility mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry of a multi-subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg(2+). We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg(2+)-dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly.

  20. Non-SMC condensin I complex, subunit D2 gene polymorphisms are associated with Parkinson's disease: a Han Chinese study.

    PubMed

    Zhang, Ping; Liu, Ling; Huang, Jinsha; Shao, Liang; Wang, Hongcai; Xiong, Nian; Wang, Tao

    2014-05-01

    Previous studies have indicated that non-SMC condensin I complex, subunit D2 (NCAPD2), an important protein in chromosome condensation, gene polymorphisms are associated with Alzheimer's disease. But no study has shown the relationship between NCAPD2 polymorphisms and Parkinson's disease. Here, we conducted a case-control study to investigate the relationship between NCAPD2 polymorphisms and the risk of Parkinson's disease in a Han Chinese population. Two single nuclear polymorphisms (SNPs) of NCAPD2 (rs7311174 and rs2072374) showed significant p values (p = 0.046 and p = 0.043, respectively) in 265 patients and 267 controls. Further analysis showed an effect of age and gender on the relationship between the two SNPs and the risk for Parkinson's disease. The A allele of rs7311174 and the T allele of rs2072374 were protective in the male patients (p = 0.016 and p = 0.019, respectively). The frequencies of the T allele of rs7311174 and the C allele of rs2072374 were significantly associated with late-onset Parkinson's disease (p = 0.048 and p = 0.044, respectively). This research demonstrates a positive relationship between the NCAPD2 gene and the risk for Parkinson's disease in a Han Chinese population and provides a potential genetic marker for sporadic Parkinson's disease.

  1. Translation initiation factor (iso) 4E interacts with BTF3, the beta subunit of the nascent polypeptide-associated complex.

    PubMed

    Freire, Miguel Angel

    2005-01-31

    A two-hybrid screen with the translation initiation factor, eIF(iso)4E from Arabidopsis, identified a clone encoding a lipoxygenase type 2 [Freire, M.A., et al., 2000. Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein. Plant Molecular Biology 44, 129-140], and three cDNA clones encoding the homologue of the mammalian BTF3 factor, the beta subunit of the nascent polypeptide-associated complex (NAC). Here we report on the interaction between the translation initiation factor eIF(iso)4E and AtBTF3. AtBTF3 protein is able to interact with the wheat initiation factors eIF4E and eIF(iso)4E. AtBTF3 contains a sequence related to the prototypic motif found on most of the 4E-binding proteins, and competes with the translation initiation factor eIF(iso)4G for eIF4(iso)4E binding, in a two hybrid interference assay. These findings provide a molecular link between the translation initiation mechanism and the emergence of the nascent polypeptide chains.

  2. Two putative subunits of a peptide pump encoded in the human major histocompatibility complex class II region.

    PubMed Central

    Bahram, S; Arnold, D; Bresnahan, M; Strominger, J L; Spies, T

    1991-01-01

    The class II region of the human major histocompatibility complex (MHC) may encode several genes controlling the processing of endogenous antigen and the presentation of peptide epitopes by MHC class I molecules to cytotoxic T lymphocytes. A previously described peptide supply factor (PSF1) is a member of the multidrug-resistance family of transporters and may pump cytosolic peptides into the membrane-bound compartment where class I molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class II DOB gene. The complete sequences of PSF1 and PSF2 were determined from cDNA clones. The translation products are closely related in sequence and predicted secondary structure. Both contain a highly conserved ATP-binding fold and share 25% homology in a hydrophobic domain with a tentative number of eight membrane-spanning segments. Based on the principle dimeric organization of these two domains in other transporters, PSF1 and PSF2 may function as complementary subunits, independently as homodimers, or both. Taken together with previous genetic evidence, the coregulation of PSF1 and PSF2 by gamma interferon and the to-some-degree coordinate transcription of these genes suggest a common role in peptide-loading of class I molecules, although a distinct function of PSF2 cannot be ruled out. Images PMID:1946428

  3. Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

    PubMed Central

    Sugimoto, K; Shimomura, T; Hashimoto, K; Araki, H; Sugino, A; Matsumoto, K

    1996-01-01

    The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692942

  4. LIN9, a subunit of the DREAM complex, regulates mitotic gene expression and proliferation of embryonic stem cells.

    PubMed

    Esterlechner, Jasmina; Reichert, Nina; Iltzsche, Fabian; Krause, Michael; Finkernagel, Florian; Gaubatz, Stefan

    2013-01-01

    The DREAM complex plays an important role in regulation of gene expression during the cell cycle. We have previously shown that the DREAM subunit LIN9 is required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this study we examined the effect of knocking down LIN9 on ESCs. We demonstrate that depletion of LIN9 alters the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. Genome-wide expression studies showed that the depletion of LIN9 results in downregulation of mitotic genes and in upregulation of differentiation-specific genes. ChIP-on chip experiments showed that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of pluripotency markers SOX2, OCT4 and Nanog and LIN9 depleted ESCs retain alkaline phosphatase activity. We conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis.

  5. Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme.

    PubMed

    Cardol, Pierre; Matagne, René F; Remacle, Claire

    2002-06-21

    The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components (ND1, ND2, ND4, ND5 and ND6) are coded for by the mitochondrial genome. Here, we characterize two mitochondrial mutants (dum5 and dum17) showing strong reduction or inactivation of complex I activity: dum5 is a 1T deletion in the 3' UTR of nd5 whereas dum17 is a 1T deletion in the coding sequence of nd6. The impact of these mutations and of mutations affecting nd1, nd4 and nd4/nd5 genes on the assembly of complex I is investigated. After separation of the respiratory complexes by blue native (BN)-PAGE or sucrose gradient centrifugation, we demonstrate that the absence of intact ND1 or ND6 subunit prevents the assembly of the 850 kDa whole complex, whereas the loss of ND4 or ND4/ND5 leads to the formation of a subcomplex of 650 kDa present in reduced amount. The implications of our findings for the possible role of these ND subunits on the activity of complex I and for the structural organization of the membrane arm of the enzyme are discussed. In mitochondria from all the strains analyzed, we moreover detected a 160-210 kDa fragment comprising the hydrophilic 49 kDa and 76 kDa subunits of the complex I peripheral arm and showing NADH dehydrogenase activity. (c) 2002 Elsevier Science Ltd.

  6. A single-molecule view of the assembly pathway, subunit stoichiometry, and unwinding activity of the bacteriophage T4 primosome (helicase-primase) complex.

    PubMed

    Lee, Wonbae; Jose, Davis; Phelps, Carey; Marcus, Andrew H; von Hippel, Peter H

    2013-05-07

    Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase-primase (primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks "internally" labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form functional and fully active primosome-DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)(6) complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting primosome complex at the fork and resulted in fully active and processive primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the primosome unwinding activity. The use of alternative assembly pathways resulted in a loss of helicase activity and the formation of metastable DNA-protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single-molecule experiments provide a detailed real-time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies.

  7. Bacterial-type Phosphoenolpyruvate Carboxylase (PEPC) Functions as a Catalytic and Regulatory Subunit of the Novel Class-2 PEPC Complex of Vascular Plants*

    PubMed Central

    O'Leary, Brendan; Rao, Srinath K.; Kim, Julia; Plaxton, William C.

    2009-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated anaplerotic enzyme situated at a major branch point of the plant C metabolism. Two distinct oligomeric classes of PEPC occur in the triglyceride-rich endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of identical 107-kDa plant-type PEPC (PTPC) subunits (encoded by RcPpc3), whereas the novel Class-2 PEPC 910-kDa hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterial-type PEPC (BTPC) polypeptides (encoded by RcPpc4). Here, COS BTPC was expressed from full-length RcPpc4 cDNA in Escherichia coli as an active PEPC that exhibited unusual properties relative to PTPCs, including a tendency to form large aggregates, enhanced thermal stability, a high Km(PEP), and insensitivity to metabolite effectors. A chimeric 900-kDa Class-2 PEPC hetero-octamer having a 1:1 stoichiometry of BTPC:PTPC subunits was isolated from a mixture of clarified extracts containing recombinant RcPPC4 and an Arabidopsis thaliana Class-1 PEPC (the PTPC, AtPPC3). The purified Class-2 PEPC exhibited biphasic PEP saturation kinetics with high and low affinity sites attributed to its AtPPC3 and RcPPC4 subunits, respectively. The RcPPC4 subunits: (i) catalyzed the majority of the Class-2 PEPC Vmax, particularly in the presence of the inhibitor l-malate, and (ii) also functioned as Class-2 PEPC regulatory subunits by modulating PEP binding and catalytic potential of its AtPPC3 subunits. BTPCs appear to associate with PTPCs to form stable Class-2 PEPC complexes in vivo that are hypothesized to maintain high flux from PEP under physiological conditions that would otherwise inhibit Class-1 PEPCs. PMID:19605358

  8. Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic and regulatory subunit of the novel class-2 PEPC complex of vascular plants.

    PubMed

    O'Leary, Brendan; Rao, Srinath K; Kim, Julia; Plaxton, William C

    2009-09-11

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated anaplerotic enzyme situated at a major branch point of the plant C metabolism. Two distinct oligomeric classes of PEPC occur in the triglyceride-rich endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of identical 107-kDa plant-type PEPC (PTPC) subunits (encoded by RcPpc3), whereas the novel Class-2 PEPC 910-kDa hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterial-type PEPC (BTPC) polypeptides (encoded by RcPpc4). Here, COS BTPC was expressed from full-length RcPpc4 cDNA in Escherichia coli as an active PEPC that exhibited unusual properties relative to PTPCs, including a tendency to form large aggregates, enhanced thermal stability, a high K(m)((PEP)), and insensitivity to metabolite effectors. A chimeric 900-kDa Class-2 PEPC hetero-octamer having a 1:1 stoichiometry of BTPC:PTPC subunits was isolated from a mixture of clarified extracts containing recombinant RcPPC4 and an Arabidopsis thaliana Class-1 PEPC (the PTPC, AtPPC3). The purified Class-2 PEPC exhibited biphasic PEP saturation kinetics with high and low affinity sites attributed to its AtPPC3 and RcPPC4 subunits, respectively. The RcPPC4 subunits: (i) catalyzed the majority of the Class-2 PEPC V(max), particularly in the presence of the inhibitor l-malate, and (ii) also functioned as Class-2 PEPC regulatory subunits by modulating PEP binding and catalytic potential of its AtPPC3 subunits. BTPCs appear to associate with PTPCs to form stable Class-2 PEPC complexes in vivo that are hypothesized to maintain high flux from PEP under physiological conditions that would otherwise inhibit Class-1 PEPCs.

  9. Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase.

    PubMed

    Sun, Y; Conner, J

    2000-04-01

    We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction.

  10. Electrochemiluminescence aptasensor for adenosine triphosphate detection using host-guest recognition between metallocyclodextrin complex and aptamer.

    PubMed

    Chen, Hong; Chen, Qiong; Zhao, Yingying; Zhang, Fan; Yang, Fan; Tang, Jie; He, Pingang

    2014-04-01

    A sensitive and label-free electrochemiluminescence (ECL) aptasensor for the detection of adenosine triphosphate (ATP) was successfully designed using host-guest recognition between a metallocyclodextrin complex, i.e., tris(bipyridine)ruthenium(II)-β-cyclodextrin [tris(bpyRu)-β-CD], and an ATP-binding aptamer. In the protocol, the NH2-terminated aptamer was immobilized on a glassy carbon electrode (GCE) by a coupling interaction. After host-guest recognition between tris(bpyRu)-β-CD and aptamer, the tris(bpyRu)-β-CD/aptamer/GCE produced a strong ECL signal as a result of the photoactive properties of tris(bpyRu)-β-CD. However, in the presence of ATP, the ATP/aptamer complex was formed preferentially, which restricted host-guest recognition, and therefore less tris(bpyRu)-β-CD was attached to the GCE surface, resulting in an obvious decrease in the ECL intensity. Under optimal determination conditions, an excellent logarithmic linear relationship between the ECL decrease and ATP concentration was obtained in the range 10.0-0.05 nM, with a detection limit of 0.01 nM at the S/N ratio of 3. The proposed ECL-based ATP aptasensor exhibited high sensitivity and selectivity, without time-consuming signal-labeling procedures, and is considered to be a promising model for detection of aptamer-specific targets.

  11. β-Catenin–related protein WRM-1 is a multifunctional regulatory subunit of the LIT-1 MAPK complex

    PubMed Central

    Yang, Xiao-Dong; Karhadkar, Tejas R.; Medina, Jessica; Robertson, Scott M.; Lin, Rueyling

    2015-01-01

    Vertebrate β-catenin has two functions, as a structural component of the adherens junction in cell adhesion and as the T-cell factor (TCF) transcriptional coactivator in canonical Wnt (wingless-related integration site) signaling. These two functions are split between three of the four β-catenin–related proteins present in the round worm Caenorhabditis elegans. The fourth β-catenin–related protein, WRM-1, exhibits neither of these functions. Instead, WRM-1 binds the MAPK loss of intestine 1 (LIT-1), and these two proteins have been shown to be essential for the transcription of Wnt target genes by phosphorylating and regulating the nuclear level of the sole worm TCF protein. We showed previously that WRM-1 binds to worm TCF and functions as the substrate-binding subunit for LIT-1. In this study, we show that phosphorylation of T220 in the activation loop is essential for LIT-1 kinase activity in vivo and in vitro. T220 can be phosphorylated either through LIT-1 autophosphorylation or directly by the upstream MAP3K MOM-4. Our data support a model in which WRM-1, which can undergo homotypic interaction, binds LIT-1 and thereby generates a kinase complex in which LIT-1 molecules are situated in a conformation enabling autophosphorylation as well as promoting phosphorylation of the T220 residue by MOM-4. In addition, we show that WRM-1 is essential for the translocation of the LIT-1 kinase complex to the nucleus, the site of its TCF substrate. To our knowledge, this is the first report of a MAP3K directly activating a MAPK by phosphorylation within the activation loop. This study should help uncover novel and as yet underappreciated functions of vertebrate β-catenin. PMID:25548171

  12. REF4 and RFR1, Subunits of the Transcriptional Coregulatory Complex Mediator, Are Required for Phenylpropanoid Homeostasis in Arabidopsis*

    PubMed Central

    Bonawitz, Nicholas D.; Soltau, Whitney L.; Blatchley, Michael R.; Powers, Brendan L.; Hurlock, Anna K.; Seals, Leslie A.; Weng, Jing-Ke; Stout, Jake; Chapple, Clint

    2012-01-01

    The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs. PMID:22167189

  13. Increased Mobility of Major Histocompatibility Complex I-Peptide Complexes Decreases the Sensitivity of Antigen Recognition*S⃞

    PubMed Central

    Segura, Jean-Manuel; Guillaume, Philippe; Mark, Silke; Dojcinovic, Danijel; Johannsen, Alexandre; Bosshard, Giovanna; Angelov, Georgi; Legler, Daniel F.; Vogel, Horst; Luescher, Immanuel F.

    2008-01-01

    CD8+ cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2Kd molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules. PMID:18579518

  14. Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants

    PubMed Central

    Gutierres, Sophie; Sabar, Mohammed; Lelandais, Christine; Chetrit, Philippe; Diolez, Philippe; Degand, Hervé; Boutry, Marc; Vedel, Fernand; de Kouchkovsky, Yaroslav; De Paepe, Rosine

    1997-01-01

    We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection. PMID:9096412

  15. The chromatin remodeling and mRNA splicing functions of the Brahma (SWI/SNF) complex are mediated by the SNR1/SNF5 regulatory subunit.

    PubMed

    Zraly, Claudia B; Dingwall, Andrew K

    2012-07-01

    Nucleosome remodeling catalyzed by the ATP-dependent SWI/SNF complex is essential for regulated gene expression. Transcriptome profiling studies in flies and mammals identified cell cycle and hormone responsive genes as important targets of remodeling complex activities. Loss of chromatin remodeling function has been linked to developmental abnormalities and aggressive cancers. The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development. We used RNAi knockdown in cultured cells and transgenic flies, and conditional mutant alleles to identify unique and important functions of two conserved Brm complex core subunits, SNR1/SNF5 and BRM/SNF2-SWI2, on target gene regulation. Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region. Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.

  16. The VPS-20 subunit of the endosomal sorting complex ESCRT-III exhibits an open conformation in the absence of upstream activation.

    PubMed

    Schuh, Amber L; Hanna, Michael; Quinney, Kyle; Wang, Lei; Sarkeshik, Ali; Yates, John R; Audhya, Anjon

    2015-03-15

    Members of the endosomal sorting complex required for transport (ESCRT) machinery function in membrane remodelling processes during multivesicular endosome (MVE) biogenesis, cytokinesis, retroviral budding and plasma membrane repair. During luminal vesicle formation at endosomes, the ESCRT-II complex and the ESCRT-III subunit vacuolar protein sorting (VPS)-20 play a specific role in regulating assembly of ESCRT-III filaments, which promote vesicle scission. Previous work suggests that Vps20 isoforms, like other ESCRT-III subunits, exhibits an auto-inhibited closed conformation in solution and its activation depends on an association with ESCRT-II specifically at membranes [1]. However, we show in the present study that Caenorhabditis elegans ESCRT-II and VPS-20 interact directly in solution, both in cytosolic cell extracts and in using recombinant proteins in vitro. Moreover, we demonstrate that purified VPS-20 exhibits an open extended conformation, irrespective of ESCRT-II binding, in contrast with the closed auto-inhibited architecture of another ESCRT-III subunit, VPS-24. Our data argue that individual ESCRT-III subunits adopt distinct conformations, which are tailored for their specific functions during ESCRT-mediated membrane reorganization events.

  17. Mono-nuclear copper complexes mimicking the intermediates for the binuclear copper center of the subunit II of cytochrome oxidase: a peptide based approach.

    PubMed

    Dutta Gupta, Dwaipayan; Usharani, Dandamudi; Mazumdar, Shyamalava

    2016-11-28

    Three stable copper complexes of peptides derived from the copper ion binding loop of the subunit II of cytochrome c oxidase have been prepared and characterized by various spectroscopic techniques. These stable copper complexes of peptides were found to exhibit cysteine, histidine and/or methionine ligation, which has predominant σ-contribution in the Cys-Cu charge transfer. The copper(ii) peptide complexes showed type-2 EPR spectra, which is uncommon in copper-cysteinate complexes. UV-visible spectra, Raman and EPR results support a tetragonal structure of the coordination geometry around the copper ion. The copper complex of the 9-amino acid peptide suggested the formation of a 'red' copper center while the copper complexes of the 12- and 11-amino acid peptides showed the formation of a 'green' copper center. The results provide insights on the first stable models of the copper complexes formed in the peptide scaffold that mimic the mono-nuclear copper bound protein intermediates proposed during the formation of the binuclear Cu2S2 core of the enzyme. These three copper complexes of peptides derived from the metal ion binding loop of the CuA center of the subunit II of cytochrome c oxidase showed novel spectroscopic properties which have not so far been reported in any stable small complex.

  18. Analysis of the subunit organization of the eIF2B complex reveals new insights into its structure and regulation.

    PubMed

    Wortham, Noel C; Martinez, Magdalena; Gordiyenko, Yuliya; Robinson, Carol V; Proud, Christopher G

    2014-05-01

    Eukaryotic initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for eIF2 and a critical regulator of protein synthesis, (e.g., as part of the integrated stress response). Certain mutations in the EIF2B genes cause leukoencephalopathy with vanishing white matter (VWM), an often serious neurological disorder. Comprising 5 subunits, α-ε (eIF2Bε being the catalytic one), eIF2B has always been considered an αβγδε heteropentamer. We have analyzed the subunit interactions within mammalian eIF2B by using a combination of mass spectrometry and in vivo studies of overexpressed complexes to gain further insight into the subunit arrangement of the complex. Our data reveal that eIF2B is actually decameric, a dimer of eIF2B(βγδε) tetramers stabilized by 2 copies of eIF2Bα. We also demonstrate a pivotal role for eIF2Bδ in the formation of eIF2B(βγδε) tetramers. eIF2B(αβγδε)2 decamers show greater binding to eIF2 than to eIF2B(βγδε) tetramers, which may underlie the increased activity of the former. We examined the levels of eIF2B subunits in a panel of different mouse tissues and identified different levels of eIF2B subunits, particularly eIF2Bα, which implies heterogeneity in the cellular proportions of eIF2B(αβγδε) and eIF2B(βγδε) complexes, with important implications for the regulation of translation in individual cell types.

  19. ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

    PubMed Central

    Babot, Marion; Labarbuta, Paola; Birch, Amanda; Kee, Sara; Fuszard, Matthew; Botting, Catherine H.; Wittig, Ilka; Heide, Heinrich; Galkin, Alexander

    2014-01-01

    An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III2 + IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover. PMID:24560811

  20. Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate

    PubMed Central

    Elustondo, P A; Nichols, M; Negoda, A; Thirumaran, A; Zakharian, E; Robertson, G S; Pavlov, E V

    2016-01-01

    Mitochondrial permeability transition pore (mPTP) opening allows free movement of ions and small molecules leading to mitochondrial membrane depolarization and ATP depletion that triggers cell death. A multi-protein complex of the mitochondrial ATP synthase has an essential role in mPTP. However, the molecular identity of the central 'pore' part of mPTP complex is not known. A highly purified fraction of mammalian mitochondria containing C-subunit of ATPase (C-subunit), calcium, inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB) forms ion channels with properties that resemble the native mPTP. We demonstrate here that amount of this channel-forming complex dramatically increases in intact mitochondria during mPTP activation. This increase is inhibited by both Cyclosporine A, an inhibitor of mPTP and Ruthenium Red, an inhibitor of the Mitochondrial Calcium Uniporter. Similar increases in the amount of complex formation occurs in areas of mouse brain damaged by ischemia-reperfusion injury. These findings suggest that calcium-induced mPTP is associated with de novo assembly of a channel comprising C-subunit, polyP and PHB. PMID:27924223

  1. Complete sequence, subunit structure, and complexes with pancreatic alpha-amylase of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

    PubMed

    Kasahara, K; Hayashi, K; Arakawa, T; Philo, J S; Wen, J; Hara, S; Yamaguchi, H

    1996-07-01

    The complete amino acid sequence of a white kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor (PHA-I), which is composed of two kinds of glycopolypeptide subunits, alpha and beta, was established by conventional methods. The polypeptide molecular weight of PHA-I determined by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that PHA-I has the subunit stoichiometry of (alpha beta)2 complex. Inhibition test of PHA-I with increasing amounts of porcine pancreatic alpha-amylase (PPA) suggested that an inactive 2:1 complex is formed between PPA and PHA-I. In fact, two complexes differing from each other in the molar ratio of PPA to PHA-I were separated by gel filtration, and molecular weight estimation by the light-scattering technique confirmed that they are complexes of PHA-I with one or two PPA molecules. The binding of PPA to PHA-I appeared to follow simple binomial statistics, suggesting that two binding sites on PHA-I are independent and of high affinity for PPA.

  2. The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

    PubMed Central

    Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui; Wong, Sui-Lam; Ng, Kenneth K. S.

    2013-01-01

    The 38-residue SBP-Tag binds to streptavidin more tightly (K d ≃ 2.5–4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-­terminal HVV peptide sequence (residues 12–14) and a C-­terminal HPQ sequence (residues 31–33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17–28) adopts a regular α-­helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1–10 and 35–38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-­residue peptide comprising residues 11–34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-­terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed. PMID:23633599

  3. The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits.

    PubMed

    Barrette-Ng, Isabelle H; Wu, Sau Ching; Tjia, Wai Mui; Wong, Sui Lam; Ng, Kenneth K S

    2013-05-01

    The 38-residue SBP-Tag binds to streptavidin more tightly (K(d) -/= 2.5-4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12-14) and a C-terminal HPQ sequence (residues 31-33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17-28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1-10 and 35-38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11-34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag-streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

  4. Electron tunneling pathways in respiratory complex I. The role of the internal water between the enzyme subunits.

    PubMed

    Hayashi, Tomoyuki; Stuchebrukhov, Alexei

    2011-09-15

    Recently, the atomistic details of the electronic wiring of seven Fe/S clusters (N3, N1b, N4, N5, N6a, N6b, N2) of respiratory complex I, along which electrons are injected into the electron transport chain, have been revealed; the tunneling pathways between the clusters and the contributing key residues were identified [1]. In this study, the sensitivity of the electron tunneling pathways to the internal water at the protein subunit boundaries is investigated by simulating tunneling pathways of N3→N1b and N6b→N2 with and without the internal water. It is found that the hydrogen bonding networks formed along the internal water can provide efficient tunneling pathways. In N3→N1b, the tunneling pathway with the internal water is drastically different with significantly shorter (3.4 Å) total tunneling distance along the trajectory. In N6b→N2, the internal water contributes to the tunneling as a bridge between N6b and 9Ile(99) with two shorter through-space jumps instead of one longer jump. The resulting enhancement of the rates of the individual electron tunneling process is two to three orders of magnitude. This study demonstrates that the tunneling pathways and tunneling rates are sensitive to the internal water, which suggests that the tunneling pathways can change dynamically due to the diffusion of the internal water, and that the efficient electron tunneling occurs at some specific optimal positions of the internal water.

  5. Speech recognition against harmonic and inharmonic complexes: Spectral dips and periodicity

    PubMed Central

    Deroche, Mickael L. D.; Culling, John F.; Chatterjee, Monita; Limb, Charles J.

    2014-01-01

    Speech recognition in a complex masker usually benefits from masker harmonicity, but there are several factors at work. The present study focused on two of them, glimpsing spectrally in between masker partials and periodicity within individual frequency channels. Using both a theoretical and an experimental approach, it is demonstrated that when inharmonic complexes are generated by jittering partials from their harmonic positions, there are better opportunities for spectral glimpsing in inharmonic than in harmonic maskers, and this difference is enhanced as fundamental frequency (F0) increases. As a result, measurements of masking level difference between the two maskers can be reduced, particularly at higher F0s. Using inharmonic maskers that offer similar glimpsing opportunity to harmonic maskers, it was found that the masking level difference between the two maskers varied little with F0, was influenced by periodicity of the first four partials, and could occur in low-, mid-, or high-frequency regions. Overall, the present results suggested that both spectral glimpsing and periodicity contribute to speech recognition under masking by harmonic complexes, and these effects seem independent from one another. PMID:24815268

  6. Molecular crowding drives active Pin1 into nonspecific complexes with endogenous proteins prior to substrate recognition.

    PubMed

    Luh, Laura M; Hänsel, Robert; Löhr, Frank; Kirchner, Donata K; Krauskopf, Katharina; Pitzius, Susanne; Schäfer, Birgit; Tufar, Peter; Corbeski, Ivan; Güntert, Peter; Dötsch, Volker

    2013-09-18

    Proteins and nucleic acids maintain the crowded interior of a living cell and can reach concentrations in the order of 200-400 g/L which affects the physicochemical parameters of the environment, such as viscosity and hydrodynamic as well as nonspecific strong repulsive and weak attractive interactions. Dynamics, structure, and activity of macromolecules were demonstrated to be affected by these parameters. However, it remains controversially debated, which of these factors are the dominant cause for the observed alterations in vivo. In this study we investigated the globular folded peptidyl-prolyl isomerase Pin1 in Xenopus laevis oocytes and in native-like crowded oocyte extract by in-cell NMR spectroscopy. We show that active Pin1 is driven into nonspecific weak attractive interactions with intracellular proteins prior to substrate recognition. The substrate recognition site of Pin1 performs specific and nonspecific attractive interactions. Phosphorylation of the WW domain at Ser16 by PKA abrogates both substrate recognition and the nonspecific interactions with the endogenous proteins. Our results validate the hypothesis formulated by McConkey that the majority of globular folded proteins with surface charge properties close to neutral under physiological conditions reside in macromolecular complexes with other sticky proteins due to molecular crowding. In addition, we demonstrate that commonly used synthetic crowding agents like Ficoll 70 are not suitable to mimic the intracellular environment due to their incapability to simulate biologically important weak attractive interactions.

  7. Hand region extraction and gesture recognition from video stream with complex background through entropy analysis.

    PubMed

    Lee, JongShill; Lee, YoungJoo; Lee, EungHyuk; Hong, SeungHong

    2004-01-01

    Hand gesture recognition utilizing image processing relies upon recognition through markers or hand extraction by colors, and therefore is heavily restricted by the colors of clothes or skin. We propose a method to recognize band gestures extracted from images with a complex background for a more natural interface in HCI (human computer interaction). The proposed method obtains the image by subtracting one image from another sequential image, measures the entropy, separates hand region from images, tracks the hand region and recognizes hand gestures. Through entropy measurement, we have color information that has near distribution in complexion for regions that have big values and extracted hand region from input images. We could draw the hand region adaptively in variable lighting or individual differences because entropy offers color information as well as motion information at the same time. The detected contour using chain code for the hand region is extracted, and present centroidal profile method that is improved little more and recognized gesture of hand. In the experimental results for 6 kinds of hand gesture, it shows the recognition rate with more than 95% for person and 90-100% for each gesture at 5 frames/sec.

  8. Crystal structure of a human cleavage factor CFI(m)25/CFI(m)68/RNA complex provides an insight into poly(A) site recognition and RNA looping.

    PubMed

    Yang, Qin; Coseno, Molly; Gilmartin, Gregory M; Doublié, Sylvie

    2011-03-09

    Cleavage factor I(m) (CFI(m)) is a highly conserved component of the eukaryotic mRNA 3' processing machinery that functions in sequence-specific poly(A) site recognition through the collaboration of a 25 kDa subunit containing a Nudix domain and a larger subunit of 59, 68, or 72 kDa containing an RNA recognition motif (RRM). Our previous work demonstrated that CFI(m)25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here, we report the crystal structure of CFI(m)25 complexed with the RRM domain of CFI(m)68 and RNA. The CFI(m)25 dimer is clasped on opposite sides by two CFI(m)68 RRM domains. Each CFI(m)25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFI(m)68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFI(m) to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.

  9. The Mediator Complex Subunits MED14, MED15, and MED16 Are Involved in Defense Signaling Crosstalk in Arabidopsis

    PubMed Central

    Wang, Chenggang; Du, Xuezhu; Mou, Zhonglin

    2016-01-01

    Mediator is a highly conserved protein complex that functions as a transcriptional coactivator in RNA polymerase II (RNAPII)-mediated transcription. The Arabidopsis Mediator complex has recently been implicated in plant immune responses. Here, we compared salicylic acid (SA)-, methyl jasmonate (MeJA)-, and the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC)-induced defense and/or wound-responsive gene expression in 14 Arabidopsis Mediator subunit mutants. Our results show that MED14, MED15, and MED16 are required for SA-activated expression of the defense marker gene PATHOEGNESIS-RELATED GENE1, MED25 is required for MeJA-induced expression of the wound-responsive marker gene VEGATATIVE STORAGE PROTEIN1 (VSP1), MED8, MED14, MED15, MED16, MED18, MED20a, MED25, MED31, and MED33A/B (MED33a and MED33B) are required for MeJA-induced expression of the defense maker gene PLANT DEFENSIN1.2 (PDF1.2), and MED8, MED14, MED15, MED16, MED25, and MED33A/B are also required for ACC-triggered expression of PDF1.2. Furthermore, we investigated the involvement of MED14, MED15, and MED16 in plant defense signaling crosstalk and found that MED14, MED15, and MED16 are required for SA- and ET-mediated suppression of MeJA-induced VSP1 expression. This result suggests that MED14, MED15, and MED16 not only relay defense signaling from the SA and JA/ET defense pathways to the RNAPII transcription machinery, but also fine-tune defense signaling crosstalk. Finally, we show that MED33A/B contributes to the necrotrophic fungal pathogen Botrytis cinerea-induced expression of the defense genes PDF1.2, HEVEIN-LIKE, and BASIC CHITINASE and is required for full-scale basal resistance to B. cinerea, demonstrating a positive role for MED33 in plant immunity against necrotrophic fungal pathogens. PMID:28066497

  10. The Mediator Complex Subunits MED14, MED15, and MED16 Are Involved in Defense Signaling Crosstalk in Arabidopsis.

    PubMed

    Wang, Chenggang; Du, Xuezhu; Mou, Zhonglin

    2016-01-01

    Mediator is a highly conserved protein complex that functions as a transcriptional coactivator in RNA polymerase II (RNAPII)-mediated transcription. The Arabidopsis Mediator complex has recently been implicated in plant immune responses. Here, we compared salicylic acid (SA)-, methyl jasmonate (MeJA)-, and the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC)-induced defense and/or wound-responsive gene expression in 14 Arabidopsis Mediator subunit mutants. Our results show that MED14, MED15, and MED16 are required for SA-activated expression of the defense marker gene PATHOEGNESIS-RELATED GENE1, MED25 is required for MeJA-induced expression of the wound-responsive marker gene VEGATATIVE STORAGE PROTEIN1 (VSP1), MED8, MED14, MED15, MED16, MED18, MED20a, MED25, MED31, and MED33A/B (MED33a and MED33B) are required for MeJA-induced expression of the defense maker gene PLANT DEFENSIN1.2 (PDF1.2), and MED8, MED14, MED15, MED16, MED25, and MED33A/B are also required for ACC-triggered expression of PDF1.2. Furthermore, we investigated the involvement of MED14, MED15, and MED16 in plant defense signaling crosstalk and found that MED14, MED15, and MED16 are required for SA- and ET-mediated suppression of MeJA-induced VSP1 expression. This result suggests that MED14, MED15, and MED16 not only relay defense signaling from the SA and JA/ET defense pathways to the RNAPII transcription machinery, but also fine-tune defense signaling crosstalk. Finally, we show that MED33A/B contributes to the necrotrophic fungal pathogen Botrytis cinerea-induced expression of the defense genes PDF1.2, HEVEIN-LIKE, and BASIC CHITINASE and is required for full-scale basal resistance to B. cinerea, demonstrating a positive role for MED33 in plant immunity against necrotrophic fungal pathogens.

  11. Colorimetric and luminescent bifunctional iridium(III) complexes for the sensitive recognition of cyanide ions

    NASA Astrophysics Data System (ADS)

    Chen, Xiudan; Wang, Huili; Li, Jing; Hu, Wenqin; Li, Mei-Jin

    2017-02-01

    Two new cyclometalated iridium(III) complexes [(ppy)2Irppz]Cl (1) and [(ppy)2Irbppz]Cl (2) (where ppy = 2-phenylpyridine, ppz = 4,7-phenanthrolino-5,6:5,6-pyrazine, bppz = 2.3-di-2-pyridylpyrazine), were designed and synthesized. The structure of [(ppy)2Irppz]Cl was determined by single crystal X-ray diffraction. Their photophysical properties were also studied. This kind of complexes could coordinate with Cu2 +, the photoluminescence (PL) of the complex was quenched, and the color changed from orange-red to green. The forming M-Cu (M: complexes 1 and 2) ensemble could be further utilized as a colorimetric and emission "turn-on" bifunctional detection for CN-, especially for complex 1-Cu2 + showed a high sensitivity toward CN- with a limit of diction is 97 nM. Importantly, this kind of iridium(III) complexes shows a unique recognition of cyanide ions over other anions which makes it an eligible sensing probe for cyanide ions.

  12. NdhP is an exclusive subunit of large complex of NADPH dehydrogenase essential to stabilize the complex in Synechocystis sp. strain PCC 6803.

    PubMed

    Zhang, Jingsong; Gao, Fudan; Zhao, Jiaohong; Ogawa, Teruo; Wang, Quanxi; Ma, Weimin

    2014-07-04

    Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1 and NdhF1, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, cyclic electron transport around photosystem I, and CO2 acquisition. Two mutants sensitive to high light for growth and impaired in NDH-1-mediated cyclic electron transfer were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in sml0013 encoding NdhP, a single transmembrane small subunit of the NDH-1 complex. During prolonged incubation of the wild type thylakoid membrane with n-dodecyl β-d-maltoside (DM), about half of the NDH-1L was disassembled to NDH-1M and the rest decomposed completely without forming NDH-1M. In the ndhP deletion mutant (ΔndhP), disassembling of NDH-1L to NDH-1M occurred even on ice, and decomposition to a small piece occurred at room temperature much faster than in the wild type. Deletion of the C-terminal tail of NdhP gave the same result. The C terminus of NdhP was tagged by YFP-His6. Blue native gel electrophoresis of the DM-treated thylakoid membrane of this strain and Western analysis using the antibody against GFP revealed that NdhP-YFP-His6 was exclusively confined to NDH-1L. During prolonged incubation of the thylakoid membrane of the tagged strain with DM at room temperature, NDH-1L was partially disassembled to NDH-1M and the 160-kDa band containing NdhP-YFP-His6 and possibly NdhD1 and NdhF1. We therefore conclude that NdhP, especially its C-terminal tail, is essential to assemble NdhD1 and NdhF1 and stabilize the NDH-1L complex. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

    PubMed Central

    Bammert, Lukas; Jonas, Stefanie; Ungricht, Rosemarie; Kutay, Ulrike

    2016-01-01

    Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S ribosomal subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5′ETS and ITS1 regions of the ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S ribosomal subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth. PMID:27599843

  14. Biochemical mapping of interactions within the intraflagellar transport (IFT) B core complex: IFT52 binds directly to four other IFT-B subunits.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Vetter, Melanie; Morawetz, Michaela; Lorentzen, Esben

    2011-07-29

    Cilia and flagella are complex structures emanating from the surface of most eukaroytic cells and serve important functions including motility, signaling, and sensory reception. A process called intraflagellar transport (IFT) is of central importance to ciliary assembly and maintenance. The IFT complex is required for this transport and consists of two distinct multisubunit subcomplexes, IFT-A and IFT-B. Despite the importance of the IFT complex, little is known about its overall architecture. This paper presents a biochemical dissection of the molecular interactions within the IFT-B core complex. Two stable subcomplexes consisting of IFT88/70/52/46 and IFT81/74/27/25 were recombinantly co-expressed and purified. We identify a novel interaction between IFT70/52 and map the interaction domains between IFT52 and the other subunits within the IFT88/70/52/46 complex. Additionally, we show that IFT52 binds directly to the IFT81/74/27/25 complex, indicating that it could mediate the interaction between the two subcomplexes. Our data lead to an improved architectural map for the IFT-B core complex with new interactions as well as domain resolution mapping for several subunits.

  15. Metal complexes of chiral pentaazacrowns as conformational templates for β-turn recognition

    NASA Astrophysics Data System (ADS)

    Reaka, Andrea J. H.; Ho, Chris M. W.; Marshall, Garland R.

    2002-08-01

    Examples of reverse turns as recognition motifs in biological systems can be found in high-resolution crystal structures of antibody-peptide complexes. Development of peptidomimetics is often based on replacing the amide backbone of peptides by sugar rings, steroids, benzodiazepines, or other hetero- and carbocycles. In this approach, the chemical scaffold of the peptide backbone can be replaced while retaining activity as long as the pharmacophoric groups of the peptide side chains stay in relatively the same place; in other words, similar functional groups must overlap in space for interaction with critical receptor sites. This study evaluates the potential of metal complexes of chiral pentaazacrowns (PAC) derived by reduction of cyclic pentapeptides as β-turn mimetics. Due to the limited flexibility of the pendant chiral side groups in these metal complexes, one can potentially elicit information about the receptor-bound conformation from their binding affinities. 11 PAC crystal structures with different substitution patterns complexed with 3 different metals (Mn, Fe, Cd) as a prototypical database of potential side-chain orientations. Complexation with different metals induces subtle differences in the conformations of a particular azacrown scaffold. The lack of parameterization of transition metals for force field calculations precludes a thorough theoretical study. Thus, this study utilizes a simple geometrical comparison between the experimental data for crystalline PAC complexes and the side-chain orientations seen in classic β-turns. The FOUNDATION program was used to overlap the Cα-Cβ vectors of the corresponding ideal β-turn side-chains to all possible leaving groups of the PAC complexes. When comparing the relative orientations of the chiral side chains, a strong overlap of the bonds (between about 0.1 Å to about 0.5 Å RMS for 3 residues and up to about 1 Å RMS for 4 residues) was observed for many of the molecules. Such metal complexes may lack

  16. Metal complexes of chiral pentaazacrowns as conformational templates for beta-turn recognition.

    PubMed

    Reaka, Andrea J H; Ho, Chris M W; Marshall, Garland R

    2002-01-01

    Examples of reverse turns as recognition motifs in biological systems can be found in high-resolution crystal structures of antibody-peptide complexes. Development of peptidomimetics is often based on replacing the amide backbone of peptides by sugar rings, steroids, benzodiazepines, or other hetero- and carbocycles. In this approach, the chemical scaffold of the peptide backbone can be replaced while retaining activity as long as the pharmacophoric groups of the peptide side chains stay in relatively the same place; in other words, similar functional groups must overlap in space for interaction with critical receptor sites. This study evaluates the potential of metal complexes of chiral pentaazacrowns (PAC) derived by reduction of cyclic pentapeptides as beta-turn mimetics. Due to the limited flexibility of the pendant chiral side groups in these metal complexes, one can potentially elicit information about the receptor-bound conformation from their binding affinities. 11 PAC crystal structures with different substitution patterns complexed with 3 different metals (Mn, Fe, Cd) as a prototypical database of potential side-chain orientations. Complexation with different metals induces subtle differences in the conformations of a particular azacrown scaffold. The lack of parameterization of transition metals for force field calculations precludes a thorough theoretical study. Thus, this study utilizes a simple geometrical comparison between the experimental data for crystalline PAC complexes and the side-chain orientations seen in classic beta-turns. The FOUNDATION program was used to overlap the Calpha-Cbeta vectors of the corresponding ideal beta-turn side-chains to all possible leaving groups of the PAC complexes. When comparing the relative orientations of the chiral side chains, a strong overlap of the bonds (between about 0.1 A to about 0.5 A RMS for 3 residues and up to about 1 A RMS for 4 residues) was observed for many of the molecules. Such metal complexes

  17. Enantiomeric self-recognition in homo- and heterodinuclear macrocyclic lanthanide(III) complexes.

    PubMed

    Lisowski, Jerzy

    2011-06-20

    The controlled formation of lanthanide(III) dinuclear μ-hydroxo-bridged [Ln(2)L(2)(μ-OH)(2)X(2)](n+) complexes (where X = H(2)O, NO(3)(-), or Cl(-)) of the enantiopure chiral macrocycle L is reported. The (1)H and (13)C NMR resonances of these complexes have been assigned on the basis of COSY, NOESY, TOCSY, and HMQC spectra. The observed NOE connectivities confirm that the dimeric solid-state structure is retained in solution. The enantiomeric nature of the obtained chiral complexes and binding of hydroxide anions are reflected in their CD spectra. The formation of the dimeric complexes is accompanied by a complete enantiomeric self-recognition of the chiral macrocyclic units. The reaction of NaOH with a mixture of two different mononuclear lanthanide(III) complexes, [Ln(1)L](3+) and [Ln(2)L](3+), results in formation of the heterodinuclear [Ln(1)Ln(2)L(2)(μ-OH)(2)X(2)](n+) complexes as well as the corresponding homodinuclear complexes. The formation of the heterodinuclear complex is directly confirmed by the NOESY spectra of [EuLuL(2)(μ-OH)(2)(H(2)O)(2)](4+), which reveal close contacts between the macrocyclic unit containing the Eu(III) ion and the macrocyclic unit containing the Lu(III) ion. While the relative amounts of homo- and heterodinuclear complexes are statistical for the two lanthanide(III) ions of similar radii, a clear preference for the formation of heterodinuclear species is observed when the two mononuclear complexes contain lanthanide(III) ions of markedly different sizes, e.g., La(III) and Yb(III). The formation of heterodinuclear complexes is accompanied by the self-sorting of the chiral macrocyclic units based on their chirality. The reactions of NaOH with a pair of homochiral or racemic mononuclear complexes, [Ln(1)L(RRRR)](3+)/[Ln(2)L(RRRR)](3+), [Ln(1)L(SSSS)](3+)/[Ln(2)L(SSSS)](3+), or [Ln(1)L(rac)](3+)/[Ln(2)L(rac)](3+), results in mixtures of homochiral, homodinuclear and homochiral, heterodinuclear complexes. On the contrary, no

  18. Schizosaccharomyces pombe Arc3 is a conserved subunit of the Arp2/3 complex required for polarity, actin organization, and endocytosis

    PubMed Central

    Cabrera, Rodrigo; Suo, Jinfeng; Young, Evelin; Chang, Eric C.

    2011-01-01

    We characterized the Schizosaccharomyces pombe arc3 gene, whose product shares sequence homology with that of the budding yeast ARC18 and human ARPC3/p21 subunits of the Arp2/3 complex. Our data showed that Arc3p co-localizes with F-actin patches at the cell ends, but not with F-actin cables or the equatorial actin ring, and binds other subunits of the Arp2/3 complex. Gene deletion analysis showed that arc3 is essential for viability. When arc3 expression was repressed, F-actin patches became dispersed throughout the cell with greatly reduced mobility. Furthermore in arc3-repressed cells, endocytosis was also inhibited. Human ARPC3 rescued the viability of the S. pombe arc3 null mutant; in addition, ARPC3 also localizes to F-actin patches in human cells. These data suggest that Arc3p is an evolutionarily conserved subunit of the Arp2/3 complex required for proper F-actin organization and efficient endocytosis. PMID:21449051

  19. The Old and New Testaments of gene regulation. Evolution of multi-subunit RNA polymerases and co-evolution of eukaryote complexity with the RNAP II CTD.

    PubMed

    Burton, Zachary F

    2014-01-01

    I relate a story of genesis told from the point of view of multi-subunit RNA polymerases (RNAPs) including an Old Testament (core RNAP motifs in all cellular life) and a New Testament (the RNAP II heptad repeat carboxy terminal domain (CTD) and CTD interactome in eukarya). The Old Testament: at their active site, one class of eukaryotic interfering RNAP and ubiquitous multi-subunit RNAPs each have two-double psi β barrel (DPBB) motifs (a distinct pattern for compact 6-β sheet barrels). Between β sheets 2 and 3 of the β subunit type DPBB of all multi-subunit RNAPs is a sandwich barrel hybrid motif (SBHM) that interacts with conserved initiation and elongation factors required to utilize a DNA template. Analysis of RNAP core protein motifs, therefore, indicates that RNAP evolution can be traced from the RNA-protein world to LUCA (the last universal common ancestor) branching to LECA (the last eukaryotic common ancestor) and to the present day, spanning about 4 billion years. The New Testament: in the eukaryotic lineage, I posit that splitting RNAP functions into RNAPs I, II and III and innovations developed around the CTD heptad repeat of RNAP II and the extensive CTD interactome helps to describe how greater structural, cell cycle, epigenetic and signaling complexity co-evolved in eukaryotes relative to eubacteria and archaea.

  20. Conformational Changes in the Endosomal Sorting Complex Required for the Transport III Subunit Ist1 Lead to Distinct Modes of ATPase Vps4 Regulation.

    PubMed

    Tan, Jason; Davies, Brian A; Payne, Johanna A; Benson, Linda M; Katzmann, David J

    2015-12-11

    Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III (ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.

  1. Recognition of complex mental states in patients with alcoholism after long-term abstinence.

    PubMed

    Mátyássy, Adrienne; Kelemen, Oguz; Sárközi, Zsuzsa; Janka, Zoltán; Kéri, Szabolcs

    2006-01-01

    Previous studies demonstrated that patients with alcoholism display impaired emotional facial expression recognition even after long-term abstinence. These studies focused on basic emotions (happiness, anger, sadness, and disgust). In this study, we investigated the recognition of complex social emotions and mental states in patients with alcoholism after long-term abstinence and healthy control subjects. Thirty patients with DSM-IV alcohol dependence and 30 age-matched, gender-matched, education-matched, and IQ-matched healthy control subjects participated. The patients were abstinent for >6 months. For the assessment of the recognition of complex social emotions and mental states, the Baron-Cohen Eyes Test was used. The experimenter presented 29 photographs of the eye-region of faces of actors and actresses on separate cards. Participants were asked to choose which of the four words (one target and three foils) best described the mental state of the actor/actress (for example, interested, doubtful, flirtatious, and insisting). The primary dependent measure was the number of correctly recognized stimuli. Patients with alcoholism correctly identified 22.4 (SD = 3.4) stimuli, whereas control participants identified 22.5 (SD = 2.9) stimuli. The difference was not statistically significant (P = 0.85). There was no significant difference in the proportion of patients and controls who correctly recognized each mental state. These results are against the hypotheses suggesting long-term adverse effects of alcohol on social cognition or supposing an inherent vulnerability of patients that may manifest before the development of alcohol dependence.

  2. Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues.

    PubMed

    Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia; Calzia, Daniela; Beitia, Maider; Morelli, Alessandro; Rosano, Camillo

    2017-01-22

    F1Fo-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H(+) flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H(+) within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F1 and FO domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Unique and Interactive Associations Between Maltreatment and Complex Emotion Recognition Deficits and Psychopathic Traits in an Undergraduate Sample.

    PubMed

    Waller, Rebecca; McCabe, Hannah K; Dotterer, Hailey L; Neumann, Craig S; Hyde, Luke W

    2017-09-13

    Psychopathy is defined by affective and interpersonal deficits, deviant lifestyle, and antisocial behaviors. Poor recognition of emotions and childhood maltreatment are two risk factors implicated in psychopathy. The current study examined whether childhood maltreatment and complex emotion recognition deficits showed unique and interactive associations with psychopathic traits among 261 undergraduate students. Results indicate that maltreatment was related to higher general psychopathy scores within a bifactor model comprising a general psychopathy factor and four specific factors tapping underlying dimensions of psychopathy (i.e., affective, interpersonal, lifestyle, and antisocial). A significant interaction emerged whereby maltreatment was related to higher antisocial factor scores among individuals showing poor recognition of positive emotions. In an intriguing interaction, more maltreatment was related to lower interpersonal factor scores among individuals with low/mean levels of neutral emotion recognition. The interaction of positive emotion recognition deficits and maltreatment highlights a potential intervention target among antisocial individuals who have experienced maltreatment.

  4. Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen

    PubMed Central

    Chang, Y. Paul; Xu, Meng; Machado, Ana Carolina Dantas; Yu, Xian Jessica; Rohs, Remo; Chen, Xiaojiang S.

    2013-01-01

    SUMMARY The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/ helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. PMID:23545501

  5. Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus[C][W

    PubMed Central

    Rexroth, Sascha; Rexroth, Dorothea; Veit, Sebastian; Plohnke, Nicole; Cormann, Kai U.; Nowaczyk, Marc M.; Rögner, Matthias

    2014-01-01

    The cyanobacterial cytochrome b6f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b6f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700+ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b6f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b6f complexes with different functions that might be correlated with supercomplex formation. PMID:25139006

  6. Functional Characterization of the Subunits N, H, J, and O of the NAD(P)H Dehydrogenase Complexes in Synechocystis sp. Strain PCC 6803.

    PubMed

    He, Zhihui; Mi, Hualing

    2016-06-01

    The cyanobacterial NAD(P)H dehydrogenase (NDH-1) complexes play crucial roles in variety of bioenergetic reactions such as respiration, CO2 uptake, and cyclic electron transport around PSI. Recently, substantial progress has been made in identifying the composition of subunits of NDH-1 complexes. However, the localization and the physiological roles of several subunits in cyanobacteria are not fully understood. Here, by constructing fully segregated ndhN, ndhO, ndhH, and ndhJ null mutants in Synechocystis sp. strain PCC 6803, we found that deletion of ndhN, ndhH, or ndhJ but not ndhO severely impaired the accumulation of the hydrophilic subunits of the NDH-1 in the thylakoid membrane, resulting in disassembly of NDH-1MS, NDH-1MS', as well as NDH-1L, finally causing the severe growth suppression phenotype. In contrast, deletion of NdhO affected the growth at pH 6.5 in air. In the cytoplasm, either NdhH or NdhJ deleted mutant, but neither NdhN nor NdhO deleted mutant, failed to accumulate the NDH-1 assembly intermediate consisting of NdhH, NdhJ, NdhK, and NdhM. Based on these results, we suggest that NdhN, NdhH, and NdhJ are essential for the stability and the activities of NDH-1 complexes, while NdhO for NDH-1 functions under the condition of inorganic carbon limitation in Synechocystis sp. strain PCC 6803. We discuss the roles of these subunits and propose a new NDH-1 model. © 2016 American Society of Plant Biologists. All Rights Reserved.

  7. Mutations in the gene encoding C8orf38 block complex I assembly by inhibiting production of the mitochondria-encoded subunit ND1.

    PubMed

    McKenzie, Matthew; Tucker, Elena J; Compton, Alison G; Lazarou, Michael; George, Christa; Thorburn, David R; Ryan, Michael T

    2011-12-02

    The assembly of complex I (NADH-ubiquinone oxidoreductase) is a complicated process, requiring the integration of 45 subunits encoded by both nuclear and mitochondrial DNAs into a structure of approximately 1 MDa. A number of "assembly factors" that aid complex I biogenesis have recently been described, including C8orf38. This protein was identified as an assembly factor by its evolutionary conservation in organisms containing complex I and by a C8orf38 mutation in a patient presenting with Leigh syndrome and isolated complex I deficiency. In this report, we have undertaken the characterization of C8orf38 and its role in complex I assembly. Analysis of mitochondria from fibroblasts of a patient harboring a C8orf38 mutation showed almost undetectable levels of steady-state complex I and defective biogenesis of the mtDNA-encoded subunit ND1. Complementation with wild-type C8orf38 restored the levels of both ND1 and complex I, confirming the C8orf38 mutation as the cause of the complex I defect in the patient. In the absence of ND1 in patient cells, early- and mid-stage intermediate complexes were still formed; however, assembly of late-stage intermediates was impaired, indicating a convergence point in the assembly process. While C8orf38 appears to behave at a step in complex I biogenesis similar to that of the assembly factor C20orf7, complementation studies showed that both proteins are required for ND1 synthesis/stabilization. We conclude that C8orf38 is a crucial factor required for the translation and/or integration of ND1 into an early-stage assembly intermediate and that mutation of C8orf38 disrupts the initial stages of complex I biogenesis.

  8. Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit, Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

    PubMed Central

    Sailasree, Purnima; Singh, Durgesh K.; Kameshwari, Duvurri B.; Shivaji, Sisinthy

    2014-01-01

    Background/Aims The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. Methodology and Principal Findings Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. Conclusions This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the

  9. The Cambridge Mindreading (CAM) Face-Voice Battery: Testing Complex Emotion Recognition in Adults with and without Asperger Syndrome

    ERIC Educational Resources Information Center

    Golan, Ofer; Baron-Cohen, Simon; Hill, Jacqueline

    2006-01-01

    Adults with Asperger Syndrome (AS) can recognise simple emotions and pass basic theory of mind tasks, but have difficulties recognising more complex emotions and mental states. This study describes a new battery of tasks, testing recognition of 20 complex emotions and mental states from faces and voices. The battery was given to males and females…

  10. The Cambridge Mindreading (CAM) Face-Voice Battery: Testing Complex Emotion Recognition in Adults with and without Asperger Syndrome

    ERIC Educational Resources Information Center

    Golan, Ofer; Baron-Cohen, Simon; Hill, Jacqueline

    2006-01-01

    Adults with Asperger Syndrome (AS) can recognise simple emotions and pass basic theory of mind tasks, but have difficulties recognising more complex emotions and mental states. This study describes a new battery of tasks, testing recognition of 20 complex emotions and mental states from faces and voices. The battery was given to males and females…

  11. Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells.

    PubMed

    Kara, Nihan; Hossain, Manzar; Prasanth, Supriya G; Stillman, Bruce

    2015-05-08

    Replication of eukaryotic chromosomes occurs once every cell division cycle in normal cells and is a tightly controlled process that ensures complete genome duplication. The origin recognition complex (ORC) plays a key role during the initiation of DNA replication. In human cells, the level of Orc1, the largest subunit of ORC, is regulated during the cell division cycle, and thus ORC is a dynamic complex. Upon S phase entry, Orc1 is ubiquitinated and targeted for destruction, with subsequent dissociation of ORC from chromosomes. Time lapse and live cell images of human cells expressing fluorescently tagged Orc1 show that Orc1 re-localizes to condensing chromatin during early mitosis and then displays different nuclear localization patterns at different times during G1 phase, remaining associated with late replicating regions of the genome in late G1 phase. The initial binding of Orc1 to mitotic chromosomes requires C-terminal amino acid sequences that are similar to mitotic chromosome-binding sequences in the transcriptional pioneer protein FOXA1. Depletion of Orc1 causes concomitant loss of the mini-chromosome maintenance (Mcm2-7) helicase proteins on chromatin. The data suggest that Orc1 acts as a nucleating center for ORC assembly and then pre-replication complex assembly by binding to mitotic chromosomes, followed by gradual removal from chromatin during the G1 phase.

  12. Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1

    PubMed Central

    Scognamiglio, Roberta; Notomista, Eugenio; Barbieri, Paola; Pucci, Piero; Piaz, Fabrizio Dal; Tramontano, Anna; Di Donato, Alberto

    2001-01-01

    A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building. PMID:11344317

  13. A minimal Tat system from a gram-positive organism: a bifunctional TatA subunit participates in discrete TatAC and TatA complexes.

    PubMed

    Barnett, James P; Eijlander, Robyn T; Kuipers, Oscar P; Robinson, Colin

    2008-02-01

    The Tat system transports folded proteins across bacterial and thylakoid membranes. In Gram-negative organisms, a TatABC substrate-binding complex and separate TatA complex are believed to coalesce to form an active translocon, with all three subunits essential for translocation. Most Gram-positive organisms lack a tatB gene, indicating major differences in organization and possible differences in mode of action. Here, we have studied Tat complexes encoded by the tatAdCd genes of Bacillus subtilis. Expression of tatAdCd in an Escherichia coli tat null mutant results in efficient export of a large, cofactor-containing E. coli Tat substrate, TorA. We show that the tatAd gene complements E. coli mutants lacking either tatAE or tatB, indicating a bifunctional role for this subunit in B. subtilis. Second, we have identified and characterized two distinct Tat complexes that are novel in key respects: a TatAdCd complex of approximately 230 kDa that is significantly smaller than the analogous E. coli TatABC complex (approximately 370 kDa on BN gels) and a separate TatAd complex. The latter is a discrete entity of approximately 270 kDa as judged by gel filtration chromatography, very different from the highly heterogeneous E. coli TatA complex that ranges in size from approximately 50 kDa to over 600 kDa. TatA heterogeneity has been linked to the varying size of Tat substrates being translocated, but the singular nature of the B. subtilis TatAd complex suggests that discrete TatAC and TatA complexes may form a single form of translocon.

  14. NMR resonance assignments for the N-terminal domain of the δ subunit of the E. coli γ clamp loader complex.

    PubMed

    Alyami, Esmael M; Rizzo, Alessandro A; Beuning, Penny J; Korzhnev, Dmitry M

    2017-03-06

    The β-clamp protein and the γ clamp loader complex are essential components of bacterial DNA replication machinery. The β-clamp is a ring-shaped homodimer that encircles DNA and increases the efficiency of replication by providing a binding platform for DNA polymerases and other replication-related proteins. The β-clamp is loaded onto DNA by the five-subunit γ clamp loader complex in a multi-step ATP-dependent process. The initial steps of this process involve the cooperative binding of the β-clamp by the five subunits of ATP-bound clamp loader, which induces or traps an open conformation of the clamp. Remarkably, the δ subunit of the E. coli clamp loader, or even its 140 residue N-terminal domain (called mini-δ), alone can shift conformational equilibrium of the β-clamp towards the open state. Here we report nearly complete backbone and side-chain (1)H, (13)C and (15)N NMR resonance assignments of mini-δ that will facilitate NMR studies of the mechanisms of β-clamp opening and its loading on DNA by the clamp loader.

  15. Nse1, Nse2, and a Novel Subunit of the Smc5-Smc6 Complex, Nse3, Play a Crucial Role in Meiosis

    PubMed Central

    Pebernard, Stephanie; McDonald, W. Hayes; Pavlova, Yelena; Yates, John R.; Boddy, Michael N.

    2004-01-01

    The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1+3) holds replicated sister chromatids together until mitosis, condensin (Smc2+4) acts in chromosome condensation, and Smc5+6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5+6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5+6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5+6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5+6 complex is important for replication fork stability. PMID:15331764

  16. The AAA(+) motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy.

    PubMed

    Lundqvist, Joakim; Elmlund, Dominika; Heldt, Dana; Deery, Evelyne; Söderberg, Christopher A G; Hansson, Mats; Warren, Martin; Al-Karadaghi, Salam

    2009-09-01

    Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA(+) superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.

  17. Proteomic analysis of the human KEOPS complex identifies C14ORF142 as a core subunit homologous to yeast Gon7

    PubMed Central

    Wan, Leo C.K.; Maisonneuve, Pierre; Szilard, Rachel K.; Lambert, Jean-Philippe; Ng, Timothy F.; Manczyk, Noah; Huang, Hao; Laister, Rob; Caudy, Amy A.; Gingras, Anne-Claude; Durocher, Daniel; Sicheri, Frank

    2017-01-01

    The KEOPS/EKC complex is a tRNA modification complex involved in the biosynthesis of N6-threonylcarbamoyladenosine (t6A), a universally conserved tRNA modification found on ANN-codon recognizing tRNAs. In archaea and eukaryotes, KEOPS is composed of OSGEP/Kae1, PRPK/Bud32, TPRKB/Cgi121 and LAGE3/Pcc1. In fungi, KEOPS contains an additional subunit, Gon7, whose orthologs outside of fungi, if existent, remain unidentified. In addition to displaying defective t6A biosynthesis, Saccharomyces cerevisiae strains harboring KEOPS mutations are compromised for telomere homeostasis, growth and transcriptional co-activation. To identify a Gon7 ortholog in multicellular eukaryotes as well as to uncover KEOPS-interacting proteins that may link t6A biosynthesis to the diverse set of KEOPS mutant phenotypes, we conducted a proteomic analysis of human KEOPS. This work identified 152 protein interactors, one of which, C14ORF142, interacted strongly with all four KEOPS subunits, suggesting that it may be a core component of human KEOPS. Further characterization of C14ORF142 revealed that it shared a number of biophysical and biochemical features with fungal Gon7, suggesting that C14ORF142 is the human ortholog of Gon7. In addition, our proteomic analysis identified specific interactors for different KEOPS subcomplexes, hinting that individual KEOPS subunits may have additional functions outside of t6A biosynthesis. PMID:27903914

  18. PDZ interaction sites in integrin alpha subunits. T14853, TIP/GIPC binds to a type I recognition sequence in alpha 6A/alpha 5 and a novel sequence in alpha 6B.

    PubMed

    Tani, T T; Mercurio, A M

    2001-09-28

    We used published peptide library data to identify PDZ recognition sequences in integrin alpha subunit cytoplasmic domains and found that the alpha(6)A and alpha(5) subunits contain a type I PDZ binding site (TSDA*) (asterisk indicates the stop codon). The alpha(6)A cytoplasmic domain was used for screening a two-hybrid library to find interacting proteins. The bulk of the captured cDNAs (60%) coded for TIP-2/GIPC, a cytoplasmic protein with one PDZ domain. The interaction of TIP-2/GIPC with different integrin subunits was tested in two-hybrid and in vitro binding assays. Surprisingly, TIP-2/GIPC bound strongly to the C terminus of both alpha(6)A and alpha(6)B, although the alpha(6)B sequence (ESYS*) is not suggestive of a PDZ binding site because of its polar C-terminal residue. For high affinity interaction with TIP-2/GIPC, at least one of the residues at positions -1 and -3 must be negatively charged. An aliphatic residue at position 0 increases the affinity of but is not required for this interaction. The alpha(5) integrin subunit also bound to TIP-2/GIPC. The alpha(6) integrin and TIP-2/GIPC co-localize in retraction fibers in carcinoma cells plated on laminin, a finding suggesting a functional interaction in vivo. Our results demonstrate that both splice variants of alpha(6) integrin contain a conserved PDZ binding site that enables interaction with TIP-2/GIPC. The binding site in alpha(6)B defines a new subclass of type I PDZ interaction site, characterized by a non-aliphatic residue at position 0.

  19. The FgNot3 Subunit of the Ccr4-Not Complex Regulates Vegetative Growth, Sporulation, and Virulence in Fusarium graminearum

    PubMed Central

    Bui, Duc-Cuong; Son, Hokyoung; Shin, Ji Young; Kim, Jin-Cheol; Kim, Hun; Choi, Gyung Ja; Lee, Yin-Won

    2016-01-01

    The Ccr4-Not complex is evolutionarily conserved and important for multiple cellular functions in eukaryotic cells. In this study, the biological roles of the FgNot3 subunit of this complex were investigated in the plant pathogenic fungus Fusarium graminearum. Deletion of FgNOT3 resulted in retarded vegetative growth, retarded spore germination, swollen hyphae, and hyper-branching. The ΔFgnot3 mutants also showed impaired sexual and asexual sporulation, decreased virulence, and reduced expression of genes related to conidiogenesis. Fgnot3 deletion mutants were sensitive to thermal stress, whereas NOT3 orthologs in other model eukaryotes are known to be required for cell wall integrity. We found that FgNot3 functions as a negative regulator of the production of secondary metabolites, including trichothecenes and zearalenone. Further functional characterization of other components of the Not module of the Ccr4-Not complex demonstrated that the module is conserved. Each subunit primarily functions within the context of a complex and might have distinct roles outside of the complex in F. graminearum. This is the first study to functionally characterize the Not module in filamentous fungi and provides novel insights into signal transduction pathways in fungal development. PMID:26799401

  20. Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis[C][W

    PubMed Central

    Olinares, Paul Dominic B.; Kim, Jitae; Davis, Jerrold I.; van Wijk, Klaas J.

    2011-01-01

    The caseinolytic protease (Clp) protease system has been expanded in plant plastids compared with its prokaryotic progenitors. The plastid Clp core protease consists of five different proteolytic ClpP proteins and four different noncatalytic ClpR proteins, with each present in one or more copies and organized in two heptameric rings. We determined the exact subunit composition and stoichiometry for the intact core and each ring. The chloroplast ClpP/R protease was affinity purified from clpr4 and clpp3 Arabidopsis thaliana null mutants complemented with C-terminal StrepII-tagged versions of CLPR4 and CLPP3, respectively. The subunit stoichiometry was determined by mass spectrometry-based absolute quantification using stable isotope-labeled proteotypic peptides generated from a synthetic gene. One heptameric ring contained ClpP3,4,5,6 in a 1:2:3:1 ratio. The other ring contained ClpP1 and ClpR1,2,3,4 in a 3:1:1:1:1 ratio, resulting in only three catalytic sites. These ClpP1/R1-4 proteins are most closely related to the two subunits of the cyanobacterial P3/R complex and the identical P:R ratio suggests conserved adaptation. Furthermore, the plant-specific C-terminal extensions of the ClpP/R subunits were not proteolytically removed upon assembly, suggesting a regulatory role in Clp chaperone interaction. These results will now allow testing ClpP/R structure–function relationships using rationale design. The quantification workflow we have designed is applicable to other protein complexes. PMID:21712416

  1. Crosslinking transcription factors to their recognition sequences with PtII complexes

    NASA Technical Reports Server (NTRS)

    Chu, B. C.; Orgel, L. E.

    1992-01-01

    We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.

  2. Toxicological evaluation of complex mixtures by pattern recognition: correlating chemical fingerprints to mutagenicity.

    PubMed Central

    Eide, Ingvar; Neverdal, Gunhild; Thorvaldsen, Bodil; Grung, Bjørn; Kvalheim, Olav M

    2002-01-01

    We describe the use of pattern recognition and multivariate regression in the assessment of complex mixtures by correlating chemical fingerprints to the mutagenicity of the mixtures. Mixtures were 20 organic extracts of exhaust particles, each containing 102-170 individual compounds such as polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, oxy-PAHs, and saturated hydrocarbons. Mixtures were characterized by full-scan GC-MS (gas chromatography-mass spectrometry). Data were resolved into peaks and spectra for individual compounds by an automated curve resolution procedure. Resolved chromatograms were integrated, resulting in a predictor matrix that was used as input to a principal component analysis to evaluate similarities between mixtures (i.e., classification). Furthermore, partial least-squares projections to latent structures were used to correlate the GC-MS data to mutagenicity, as measured in the Ames Salmonella assay (i.e., calibration). The best model (high r2 and Q2) identifies the variables that co-vary with the observed mutagenicity. These variables may subsequently be identified in more detail. Furthermore, the regression model can be used to predict mutagenicity from GC-MS chromatograms of other organic extracts. We emphasize that both chemical fingerprints as well as detailed data on composition can be used in pattern recognition. PMID:12634129

  3. Plant root exudates mediate neighbour recognition and trigger complex behavioural changes.

    PubMed

    Semchenko, Marina; Saar, Sirgi; Lepik, Anu

    2014-11-01

    Some plant species are able to distinguish between neighbours of different genetic identity and attempt to pre-empt resources through root proliferation in the presence of unrelated competitors, but avoid competition with kin. However, studies on neighbour recognition have met with some scepticism because the mechanisms by which plants identify their neighbours have remained unclear. In order to test whether root exudates could mediate neighbour recognition in plants, we performed a glasshouse experiment in which plants of Deschampsia caespitosa were subjected to root exudates collected from potential neighbours of different genetic identities, including siblings and individuals belonging to the same or a different population or species. Our results show that root exudates can carry specific information about the genetic relatedness, population origin and species identity of neighbours, and trigger different responses at the whole root system level and at the level of individual roots in direct contact with locally applied exudates. Increased root density was mainly achieved through changes in morphology rather than biomass allocation, suggesting that plants are able to limit the energetic cost of selfish behaviour. This study reveals a new level of complexity in the ability of plants to interpret and react to their surroundings. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  4. Crosslinking transcription factors to their recognition sequences with PtII complexes

    NASA Technical Reports Server (NTRS)

    Chu, B. C.; Orgel, L. E.

    1992-01-01

    We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.

  5. Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex*

    PubMed Central

    Smith, Karen T.; Sardiu, Mihaela E.; Martin-Brown, Skylar A.; Seidel, Chris; Mushegian, Arcady; Egidy, Rhonda; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

    2012-01-01

    Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration. PMID:22984288

  6. Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes.

    PubMed

    Scibek, Jeffery J; Evergren, Emma; Zahn, Stefan; Canziani, Gabriela A; Van Ryk, Donald; Chaiken, Irwin M

    2002-08-15

    To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

  7. Solid state vibrational circular dichroism towards molecular recognition: chiral metal complexes intercalated in a clay mineral.

    PubMed

    Sato, Hisako; Tamura, Kenji; Takimoto, Kazuyoshi; Yamagishi, Akihiko

    2017-09-13

    Vibrational circular dichroism (VCD) spectroscopy was applied to study chirality recognition in the interlayer space of a clay mineral. Clay intercalation compounds including two kinds of chiral molecules were prepared. Firstly a cationic metal complex, Δ- or Λ-[Ru(phen)3](2+) (phen = 1,10-phenanthroline), was ion-exchanged into sodium montmorillonite. Thereafter a neutral organic molecule, R- or S-1,1'-bi-2-naphthol (denoted as R- or S-BINOL), was co-adsorbed. The solid state VCD spectra were recorded on the hybrid compounds thus prepared. The intensity of VCD peaks in the region of 1300-1400 cm(-1), which were assigned to the bending vibrations of OH groups in BINOL, was remarkably dependent on the chirality relation between the two intercalated species. This implied that BINOL took a different conformation in response to the chirality of co-existing [Ru(phen)3](2+).

  8. The role of the chromatin assembly complex (CAF-1) and its p60 subunit (CHAF1b) in homeostasis and disease

    PubMed Central

    Volk, Andrew; Crispino, John D.

    2015-01-01

    Nucleosome assembly following DNA synthesis is critical for maintaining genomic stability. The proteins directly responsible for shuttling newly synthesized histones H3 and H4 from the cytoplasm to the assembly fork during DNA replication comprise the Chromatin Assembly Factor 1 complex (CAF-1). Whereas the diverse functions of the large (CAF-1-p150, CHAF1a) and small (RbAp48, p48) subunits of the CAF-1 complex have been well-characterized in many tissues and extend beyond histone chaperone activity, the contributions of the medium subunit (CAF-1-p60, CHAF1b) are much less well understood. Although it is known that CHAF1b has multiple functional domains (7x WD repeat domain, B-like domain, and a PEST domain), how these components come together to elicit the functions of this protein are still unclear. Here, we review the biology of the CAF-1 complex, with an emphasis on CHAF1b, including its structure, regulation, and function. In addition, we discuss the possible contributions of CHAF1b and the CAF-1 complex to human diseases. Of note, CHAF1b is located within the Down syndrome critical region (DSCR) of chromosome 21. Therefore, we also address the putative contributions of its trisomy to the various manifestations of DS. PMID:26066981

  9. Structure of a Premicellar Complex of Alkyl Sulfates with the Interfacial Binding Surfaces of 4 Subunits of Phospholipase A2✰

    PubMed Central

    Pan, Ying H.; Bahnson, Brian J.

    2010-01-01

    The properties of three discrete premicellar complexes (E1#, E2#, E3#) of pig pancreatic group-IB secreted phospholipase A2 (sPLA2) with monodisperse alkyl sulfates has been characterized [Berg, O. G., et al., Biochemistry 43, 7999–8013, 2004]. Here we have solved the 2.7 Å crystal structure of group-IB sPLA2 complexed with 12 molecules of octyl sulfate (C8S) in a form consistent with a tetrameric oligomeric that exists during the E1# phase of premicellar complexes. The alkyl tails of the C8S molecules are centered in the middle of the tetrameric cluster of sPLA2 subunits. Three of the four sPLA2 subunits also contain a C8S molecule in the active site pocket. The sulfate oxygen of a C8S ligand is complexed to the active site calcium in 3 of the 4 protein active sites. The interactions of the alkyl sulfate head group with Arg-6 and Lys-10, as well as the backbone amide of Met-20, are analogous to those observed in the previously solved sPLA2 crystal structures with bound phosphate and sulfate anions. The cluster of three anions found in the present structure is postulated to be the site for nucleating the binding of anionic amphiphiles to the interfacial surface of the protein, and therefore this binding interaction has implications for interfacial activation of the enzyme. PMID:20302975

  10. Characterization of clinically identified mutations in NDUFV1, the flavin-binding subunit of respiratory complex I, using a yeast model system

    PubMed Central

    Varghese, Febin; Atcheson, Erwan; Bridges, Hannah R.; Hirst, Judy

    2015-01-01

    Dysfunctions in mitochondrial complex I (NADH:ubiquinone oxidoreductase) are both genetically and clinically highly diverse and a major cause of human mitochondrial diseases. The genetic determinants of individual clinical cases are increasingly being described, but how these genetic defects affect complex I on the molecular and cellular level, and have different clinical consequences in different individuals, is little understood. Furthermore, without molecular-level information innocent genetic variants may be misassigned as pathogenic. Here, we have used a yeast model system (Yarrowia lipolytica) to study the molecular consequences of 16 single amino acid substitutions, classified as pathogenic, in the NDUFV1 subunit of complex I. NDUFV1 binds the flavin cofactor that oxidizes NADH and is the site of complex I-mediated reactive oxygen species production. Seven mutations caused loss of complex I expression, suggesting they are detrimental but precluding further study. In two variants complex I was fully assembled but did not contain any flavin, and four mutations led to functionally compromised enzymes. Our study provides a molecular rationale for assignment of all these variants as pathogenic. However, three variants provided complex I that was functionally equivalent to the wild-type enzyme, challenging their assignment as pathogenic. By combining structural, bioinformatic and functional data, a simple scoring system for the initial evaluation of future NDUFV1 variants is proposed. Overall, our results broaden understanding of how mutations in this centrally important core subunit of complex I affect its function and provide a basis for understanding the role of NDUFV1 mutations in mitochondrial dysfunction. PMID:26345448

  11. The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly

    PubMed Central

    Welch, Matthew D.; DePace, Angela H.; Verma, Suzie; Iwamatsu, Akihiro; Mitchison, Timothy J.

    1997-01-01

    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat–containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion. PMID:9230079

  12. Characterization of clinically identified mutations in NDUFV1, the flavin-binding subunit of respiratory complex I, using a yeast model system.

    PubMed

    Varghese, Febin; Atcheson, Erwan; Bridges, Hannah R; Hirst, Judy

    2015-11-15

    Dysfunctions in mitochondrial complex I (NADH:ubiquinone oxidoreductase) are both genetically and clinically highly diverse and a major cause of human mitochondrial diseases. The genetic determinants of individual clinical cases are increasingly being described, but how these genetic defects affect complex I on the molecular and cellular level, and have different clinical consequences in different individuals, is little understood. Furthermore, without molecular-level information innocent genetic variants may be misassigned as pathogenic. Here, we have used a yeast model system (Yarrowia lipolytica) to study the molecular consequences of 16 single amino acid substitutions, classified as pathogenic, in the NDUFV1 subunit of complex I. NDUFV1 binds the flavin cofactor that oxidizes NADH and is the site of complex I-mediated reactive oxygen species production. Seven mutations caused loss of complex I expression, suggesting they are detrimental but precluding further study. In two variants complex I was fully assembled but did not contain any flavin, and four mutations led to functionally compromised enzymes. Our study provides a molecular rationale for assignment of all these variants as pathogenic. However, three variants provided complex I that was functionally equivalent to the wild-type enzyme, challenging their assignment as pathogenic. By combining structural, bioinformatic and functional data, a simple scoring system for the initial evaluation of future NDUFV1 variants is proposed. Overall, our results broaden understanding of how mutations in this centrally important core subunit of complex I affect its function and provide a basis for understanding the role of NDUFV1 mutations in mitochondrial dysfunction. © The Author 2015. Published by Oxford University Press.

  13. CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli.

    PubMed

    Beloglazova, Natalia; Kuznedelov, Konstantin; Flick, Robert; Datsenko, Kirill A; Brown, Greg; Popovic, Ana; Lemak, Sofia; Semenova, Ekaterina; Severinov, Konstantin; Yakunin, Alexander F

    2015-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.

  14. Species delimitation and recognition in the Pediomelum megalanthum complex (Fabaceae) via multivariate morphometrics

    PubMed Central

    Egan, Ashley N.

    2015-01-01

    Abstract Pediomelum is a genus endemic to North America comprising about 26 species, including the megalanthum complex, which consists of Pediomelum megalanthum and its varieties retrorsum and megalanthum, Pediomelum mephiticum, and the recently described Pediomelum verdiense and Pediomelum pauperitense. Historically, species of the megalanthum complex have been variably recognized at the species or variety levels, dependent upon the relative importance of morphological characters as diagnostic of species. Ten quantitative morphological characters regarded as diagnostic at the species level were analyzed using multivariate morphometrics across these taxa in order to examine the discriminatory power of these characters to delineate species and to aid in species delimitation. The analyses support the recognition of Pediomelum megalanthum, Pediomelum mephiticum, and Pediomelum verdiense at the species level, Pediomelum retrorsum as a variety under Pediomelum megalanthum, and suggest the sinking of Pediomelum pauperitense into Pediomelum verdiense. The findings of the present study help quantify the power of certain characters at delimiting taxa and provide a basis for taxonomic revision of the Pediomelum megalanthum complex. PMID:25698894

  15. Structural basis for the recognition of complex-type biantennary oligosaccharides by Pterocarpus angolensis lectin.

    PubMed

    Buts, Lieven; Garcia-Pino, Abel; Imberty, Anne; Amiot, Nicolas; Boons, Geert-Jan; Beeckmans, Sonia; Versées, Wim; Wyns, Lode; Loris, Remy

    2006-06-01

    The crystal structure of Pterocarpus angolensis lectin is determined in its ligand-free state, in complex with the fucosylated biantennary complex type decasaccharide NA2F, and in complex with a series of smaller oligosaccharide constituents of NA2F. These results together with thermodynamic binding data indicate that the complete oligosaccharide binding site of the lectin consists of five subsites allowing the specific recognition of the pentasaccharide GlcNAc beta(1-2)Man alpha(1-3)[GlcNAc beta(1-2)Man alpha(1-6)]Man. The mannose on the 1-6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost identical to that of concanavalin A (con A). The core mannose and the GlcNAc beta(1-2)Man moiety on the 1-3 arm on the other hand occupy a series of subsites distinct from those of con A.

  16. Subunits of ADA-two-A-containing (ATAC) or Spt-Ada-Gcn5-acetyltrasferase (SAGA) Coactivator Complexes Enhance the Acetyltransferase Activity of GCN5.

    PubMed

    Riss, Anne; Scheer, Elisabeth; Joint, Mathilde; Trowitzsch, Simon; Berger, Imre; Tora, László

    2015-11-27

    Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT (Gcn5-related N-acetyltransferase) family of HATs. In metazoans this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 acetyltransferase) and ATAC (Ada Two A-containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, whereas in the SAGA HAT module ADA2b is present instead of ADA2a. To better understand how the activity of human (h) hGCN5 is regulated in the two related, but different, HAT complexes we carried out in vitro HAT assays. We compared the activity of hGCN5 alone with its activity when it was part of purified recombinant hATAC or hSAGA HAT modules or endogenous hATAC or hSAGA complexes using histone tail peptides and full-length histones as substrates. We demonstrated that the subunit environment of the HAT complexes into which GCN5 incorporates determines the enhancement of GCN5 activity. On histone peptides we show that all the tested GCN5-containing complexes acetylate mainly histone H3K14. Our results suggest a stronger influence of ADA2b as compared with ADA2a on the activity of GCN5. However, the lysine acetylation specificity of GCN5 on histone tails or full-length histones was not changed when incorporated in the HAT modules of ATAC or SAGA complexes. Our results thus demonstrate that the catalytic activity of GCN5 is stimulated by subunits of the ADA2a- or ADA2b-containing HAT modules and is further increased by incorporation of the distinct HAT modules in the ATAC or SAGA holo-complexes.

  17. Specific DNA recognition by the Antp homeodomain: MD simulations of specific and nonspecific complexes.

    PubMed

    Gutmanas, Aleksandras; Billeter, Martin

    2004-12-01

    Four molecular dynamics simulation trajectories of complexes between the wild-type or a mutant Antennapedia homeodomain and 2 DNA sequences were generated in order to probe the mechanisms governing the specificity of DNA recognition. The starting point was published affinity measurements showing that a single protein mutation combined with a replacement of 2 base pairs yields a new high-affinity complex, whereas the other combinations, with changes on only 1 macromolecule, exhibited lower affinity. The simulations of the 4 complexes yielded fluctuating networks of interaction. On average, these networks differ significantly, explaining the switch of affinity caused by the alterations in the macromolecules. The network of mostly hydrogen-bonding interactions involving several water molecules, which was suggested both by X-ray and NMR structures of the wild-type homeodomain and its DNA operator sequence, could be reproduced in the trajectory. More interestingly, the high-affinity complex with alterations in both the protein and the DNA yielded again a dynamic but very tight network of intermolecular interactions, however, attributing a significantly stronger role to direct hydrophobic interactions at the expense of water bridges. The other 2 homeodomain-DNA complexes, with only 1 molecule altered, show on average over the trajectories a clearly reduced number of protein-DNA interactions. The observations from these simulations suggest specific experiments and thus close the circle formed by biochemical, structural, and computational studies. The shift from a water-dominated to a more "dry" interface may prove important in the design of proteins binding DNA in a specific manner.

  18. Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: cytochrome b(6)f complex from spinach and the cyanobacterium Mastigocladus laminosus.

    PubMed

    Whitelegge, Julian P; Zhang, Huamin; Aguilera, Rodrigo; Taylor, Ross M; Cramer, William A

    2002-10-01

    Highly active cytochrome b(6)f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS+). Both size-exclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (+/-3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP(+) oxidoreductase (Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Flavonucleotide:ferredoxin reductase is a subunit of the plant cytochrome b(6)f complex. J. Biol. Chem. 276, 38159-38165). While the measured masses of PetC and PetD (18935.8 and 17311.8 Da, respectively) from spinach are consistent with the published primary structure, the measured masses of cytochrome f (31934.7 Da, PetA) and cytochrome b (24886.9 Da, PetB) modestly deviate from values calculated based upon genomic sequence and known post-translational modifications. The low molecular weight protein subunits have been sequenced using tandem mass spectrometry (MSMS) without prior cleavage. Sequences derived from the MSMS spectra of these intact membrane proteins in the range of 3.2-4.2 kDa were compared with translations of genomic DNA sequence where available. Products of the spinach chloroplast genome, PetG, PetL, and PetN, all retained their initiating formylmethionine, while the nuclear encoded PetM was cleaved after import from the cytoplasm. While the sequences of PetG and PetN revealed no discrepancy with translations of the spinach chloroplast genome, Phe was detected at position 2 of PetL. The spinach chloroplast genome reports a codon for Ser at position 2 implying the presence of a DNA sequencing error or a previously undiscovered RNA editing event. Clearly, complete annotation of genomic data requires detailed

  19. Molecular characterisation of the CD79a and CD79b subunits of the B cell receptor complex in the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii): Delayed B cell immunocompetence in marsupial neonates.

    PubMed

    Duncan, Louise; Webster, Koa; Gupta, Varun; Nair, Sham; Deane, Elizabeth

    2010-08-15

    The B cell receptor (BCR) is a multiprotein complex that is pivotal to antigen recognition and signal transduction in B cells. It consists of an antigen binding component, membrane Ig (mIg), non-covalently associated with the signaling component, a disulphide-linked heterodimer of CD79a and CD79b. In this study, the gene and corresponding cDNA for CD79a and CD79b in the gray short-tailed opossum, as well as the cDNA sequences for CD79a and CD79b in the tammar wallaby, are described. Many of the structural and functional features of CD79a and CD79b were conserved in both marsupials, including the ITAM regulatory motif in the cytoplasmic tails of both subunits. The marsupial CD79 sequences shared a high degree of amino acid identities of 76% (CD79a) and 72% (CD79b) to each other, as well as 60-61% (CD79a) and 58-59% (CD79b) with their eutherian counterparts. RT-PCR analysis of CD79a and CD79b transcripts in the immune tissues of tammar pouch young revealed CD79a transcripts in the bone marrow, cervical thymus and spleen at day 10 postpartum. CD79b transcripts were detected in the bone marrow and cervical thymus at day 10 but were not detected in the spleen until day 21 postpartum. These results suggest that a functional BCR may not be assembled until day 21 postpartum and the tammar neonate may not be capable of mounting an effective adaptive immune response until this time. The molecular information presented here will allow further investigation of the role of the CD79 subunits in marsupial B cell signaling, especially during ontogeny and disease.

  20. Targeting the NAD7 subunit to mitochondria restores a functional complex I and a wild type phenotype in the Nicotiana sylvestris CMS II mutant lacking nad7.

    PubMed

    Pineau, Bernard; Mathieu, Chantal; Gérard-Hirne, Catherine; De Paepe, Rosine; Chétrit, Philippe

    2005-07-15

    The mitochondrial DNA of the Nicotiana sylvestris CMSII mutant carries a 72-kb deletion comprising the single copy nad7 gene that encodes the NAD7 subunit of the respiratory complex I (NADH-ubiquinone oxidoreductase). CMSII plants lack rotenone-sensitive complex I activity and are impaired in physiological and phenotypical traits. To check whether these changes directly result from the deletion of nad7, we constructed CMS transgenic plants (termed as CMSnad7) carrying an edited nad7 cDNA fused to the CAMV 35S promoter and to a mitochondrial targeting sequence. The nad7 sequence was transcribed and translated and the NAD7 protein directed to mitochondria in CMSnad7 transgenic plants, which recovered both wild type morphology and growth features. Blue-native/SDS gel electrophoresis and enzymatic assays showed that, whereas fully assembled complex I was absent from CMSII mitochondria, a functional complex was present in CMSnad7 mitochondria. Furthermore, a supercomplex involving complex I and complex III was present in CMSnad7 as in the wild type. Taken together, these data demonstrate that lack of complex I in CMSII was indeed the direct consequence of the absence of nad7. Hence, NAD7 is a key element for complex assembly in plants. These results also show that allotopic expression from the nucleus can fully complement the lack of a mitochondrial-encoded complex I gene.

  1. Crystal Structures of Ricin Toxin’s Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-neutralizing Single Chain Antibodies

    PubMed Central

    Rudolph, Michael J.; Vance, David J.; Cheung, Jonah; Franklin, Matthew C.; Burshteyn, Fiana; Cassidy, Michael S.; Gary, Ebony N.; Herrera, Cristina; Shoemaker, Charles B.; Mantis, Nicholas J.

    2014-01-01

    Ricin is a Select Agent Toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins (RIPs). In this study, we determined x-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA’s RNA N-glycosidase activity or induced notable allosteric changes in the toxin’s subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines. PMID:24907552

  2. Water-mediated contacts in the trp-repressor operator complex recognition process.

    PubMed

    Wibowo, Fajar R; Rauch, Christine; Trieb, Michael; Wellenzohn, Bernd; Liedl, Klaus R

    2004-04-15

    Water-mediated contacts are known as an important recognition tool in trp-repressor operator systems. One of these contacts involves two conserved base pairs (G(6).C(-6) and A(5). T(-5)) and three amino acids (Lys 72, Ile 79, and Ala 80). To investigate the nature of these contacts, we analyzed the X-ray structure (PDB code: 1TRO) of the trp-repressor operator complex by means of molecular dynamics simulations. This X-ray structure contains two dimers that exhibit structural differences. From these two different starting structures, two 10 ns molecular dynamics simulations have been performed. Both of our simulations show an increase of water molecules in the major groove at one side of the dimer, while the other side remains unchanged compared to the X-ray structure. Though the maximum residence time of the concerned water molecules decreases with an increase of solvent at the interface, these water molecules continue to play an important role in mediating DNA-protein contacts. This is shown by new stable amino acids-DNA distances and a long water residence time compared to free DNA simulation. To maintain stability of the new contacts, the preferential water binding site on O6(G6) is extended. This extension agrees with mutation experiment data on A5 and G6, which shows different relative affinity due to mutation on these bases [A. Joachimiak, T. E. Haran, P. B. Sigler, EMBO Journal 1994, Vol. 13, No. (2) pp. 367-372]. Due to the rearrangements in the system, the phosphate of the base G6 is able to interconvert to the B(II) substate, which is not observed on the other half side of the complex. The decrease of the number of hydrogen bonds between protein and DNA backbone could be the initial step of the dissociation process of the complex, or in other words an intermediate complex conformation of the association process. Thus, we surmise that these features show the importance of water-mediated contacts in the trp-repressor operator recognition process.

  3. Molecular Basis of Histone Tail Recognition by Human TIP5 PHD Finger and Bromodomain of the Chromatin Remodeling Complex NoRC

    PubMed Central

    Tallant, Cynthia; Valentini, Erica; Fedorov, Oleg; Overvoorde, Lois; Ferguson, Fleur M.; Filippakopoulos, Panagis; Svergun, Dmitri I.; Knapp, Stefan; Ciulli, Alessio

    2015-01-01

    Summary Binding of the chromatin remodeling complex NoRC to RNA complementary to the rDNA promoter mediates transcriptional repression. TIP5, the largest subunit of NoRC, is involved in recruitment to rDNA by interactions with promoter-bound TTF-I, pRNA, and acetylation of H4K16. TIP5 domains that recognize posttranslational modifications on histones are essential for recruitment of NoRC to chromatin, but how these reader modules recognize site-specific histone tails has remained elusive. Here, we report crystal structures of PHD zinc finger and bromodomains from human TIP5 and BAZ2B in free form and bound to H3 and/or H4 histones. PHD finger functions as an independent structural module in recognizing unmodified H3 histone tails, and the bromodomain prefers H3 and H4 acetylation marks followed by a key basic residue, KacXXR. Further low-resolution analyses of PHD-bromodomain modules provide molecular insights into their trans histone tail recognition, required for nucleosome recruitment and transcriptional repression of the NoRC complex. PMID:25533489

  4. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.

    PubMed

    Welch, M D; DePace, A H; Verma, S; Iwamatsu, A; Mitchison, T J

    1997-07-28

    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determin